PRMT1 inhibitors and uses thereof

ABSTRACT

Described herein are compounds of Formula (I), pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof. Compounds described herein are useful for inhibiting PRMT1 activity. Methods of using the compounds for treating PRMT1-mediated disorders are also described.

RELATED APPLICATIONS

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. patent application U.S. Ser. No. 14/212,354,filed Mar. 14, 2014, now U.S. Pat. No. 9,023,883, which claims priorityunder 35 U.S.C. §119(e) to U.S. Provisional Patent Application, U.S.Ser. No. 61/781,052, filed Mar. 14, 2013, the entire contents of each ofwhich is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Epigenetic regulation of gene expression is an important biologicaldeterminant of protein production and cellular differentiation and playsa significant pathogenic role in a number of human diseases.

Epigenetic regulation involves heritable modification of geneticmaterial without changing its nucleotide sequence. Typically, epigeneticregulation is mediated by selective and reversible modification (e.g.,methylation) of DNA and proteins (e.g., histones) that control theconformational transition between transcriptionally active and inactivestates of chromatin. These covalent modifications can be controlled byenzymes such as methyltransferases (e.g., PRMT1), many of which areassociated with specific genetic alterations that can cause humandisease.

Disease-associated chromatin-modifying enzymes (e.g., PRMT1) play a rolein diseases such as proliferative disorders, autoimmune disorders,muscular disorders, vascular disorders, metabolic disorders, andneurological disorders. Thus, there is a need for the development ofsmall molecules that are capable of inhibiting the activity of PRMT1.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

PRMT1 is an attractive target for modulation given its role in theregulation of diverse biological processes. It has now been found thatcompounds described herein, and pharmaceutically acceptable salts andcompositions thereof, are effective as inhibitors of PRMT1. Suchcompounds have the general Formula (I):

or a pharmaceutically acceptable salt thereof, wherein X, Y, Z, R^(x),R^(y), R², R³, and R⁶ are as defined herein.

In some embodiments, pharmaceutical compositions are provided whichcomprise a compound described herein (e.g., a compound of Formula (I)),or a pharmaceutically acceptable salt thereof, and optionally apharmaceutically acceptable excipient.

In certain embodiments, compounds described herein inhibit activity ofPRMT1. In certain embodiments, methods of inhibiting PRMT1 are providedwhich comprise contacting PRMT1 with an effective amount of a compoundof Formula (I), or a pharmaceutically acceptable salt thereof. The PRMT1may be purified or crude, and may be present in a cell, tissue, or asubject. Thus, such methods encompass inhibition of PRMT1 activity bothin vitro and in vivo. In certain embodiments, the PRMT1 is wild-typePRMT1. In certain embodiments, the PRMT1 is overexpressed. In certainembodiments, the PRMT1 is a mutant. In certain embodiments, the PRMT1 isin a cell. In certain embodiments, the PRMT1 is in an animal, e.g., ahuman. In some embodiments, the PRMT1 is expressed at normal levels in asubject, but the subject would benefit from PRMT1 inhibition (e.g.,because the subject has one or more mutations in an PRMT1 substrate thatcauses an increase in methylation of the substrate with normal levels ofPRMT1). In some embodiments, the PRMT1 is in a subject known oridentified as having abnormal PRMT1 activity (e.g., overexpression). Insome embodiments, a provided compound is selective for PRMT1 over othermethyltransferases. In certain embodiments, a provided compound is atleast about 10-fold selective, at least about 20-fold selective, atleast about 30-fold selective, at least about 40-fold selective, atleast about 50-fold selective, at least about 60-fold selective, atleast about 70-fold selective, at least about 80-fold selective, atleast about 90-fold selective, or at least about 100-fold selectiverelative to one or more other methyltransferases.

In certain embodiments, methods of modulating gene expression in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, cellis in an animal, e.g., a human.

In certain embodiments, methods of modulating transcription in a cellare provided which comprise contacting a cell with an effective amountof a compound of Formula (I), or a pharmaceutically acceptable saltthereof, or a pharmaceutical composition thereof. In certainembodiments, the cell in culture in vitro. In certain embodiments, thecell is in an animal, e.g., a human.

In some embodiments, methods of treating a PRMT1-mediated disorder areprovided which comprise administering to a subject suffering from aPRMT1-mediated disorder an effective amount of a compound describedherein (e.g., a compound of Formula (I)), or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition thereof. Incertain embodiments, the PRMT1-mediated disorder is a proliferativedisorder. In certain embodiments, compounds described herein are usefulfor treating cancer. In certain embodiments, compounds described hereinare useful for treating breast cancer, prostate cancer, lung cancer,colon cancer, bladder cancer, or leukemia. In certain embodiments, thePRMT1-mediated disorder is a muscular disorder. In certain embodiments,the PRMT1-mediated disorder is an autoimmune disorder. In certainembodiments, the PRMT1-mediated disorder is a neurological disorder. Incertain embodiments, the PRMT1-mediated disorder is a vascular disorder.In certain embodiments, the PRMT1-mediated disorder is a metabolicdisorder.

Compounds described herein are also useful for the study of PRMT1 inbiological and pathological phenomena, the study of intracellular signaltransduction pathways mediated by PRMT1, and the comparative evaluationof new PRMT1 inhibitors.

This application refers to various issued patent, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference.

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The present disclosureadditionally encompasses compounds described herein as individualisomers substantially free of other isomers, and alternatively, asmixtures of various isomers.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofany compound described herein does not exclude any tautomer form.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of a carbon bya 13C- or 14C-enriched carbon are within the scope of the disclosure.Such compounds are useful, for example, as analytical tools or probes inbiological assays.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), n-propyl(C₃), isopropyl (C₃), n-butyl (C₄), tert-butyl (C₄), sec-butyl (C₄),iso-butyl (C₄), n-pentyl (C₅), 3-pentanyl (C₅), amyl (C₅), neopentyl(C₅), 3-methyl-2-butanyl (C₅), tertiary amyl (C₅), and n-hexyl (C₆).Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈)and the like. In certain embodiments, each instance of an alkyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents. In certain embodiments, the alkyl group isunsubstituted C₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, thealkyl group is substituted C₁₋₁₀ alkyl.

In some embodiments, an alkyl group is substituted with one or morehalogens. “Perhaloalkyl” is a substituted alkyl group as defined hereinwherein all of the hydrogen atoms are independently replaced by ahalogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, thealkyl moiety has 1 to 8 carbon atoms (“C₁₋₈ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 6 carbon atoms (“C₁₋₆perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 4 carbonatoms (“C₁₋₄ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 3 carbon atoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkylmoiety has 1 to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In someembodiments, all of the hydrogen atoms are replaced with fluoro. In someembodiments, all of the hydrogen atoms are replaced with chloro.Examples of perhaloalkyl groups include —CF₃, —CF₂CF₃, —CF₂CF₂CF₃,—CCl₃, —CFCl₂, —CF₂Cl, and the like.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms and one ormore carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds), andoptionally one or more triple bonds (e.g., 1, 2, 3, or 4 triple bonds)(“C₂₋₂₀ alkenyl”). In certain embodiments, alkenyl does not comprisetriple bonds. In some embodiments, an alkenyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkenyl”). In some embodiments, an alkenyl group has 2 to9 carbon atoms (“C₂₋₉ alkenyl”). In some embodiments, an alkenyl grouphas 2 to 8 carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, analkenyl group has 2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In someembodiments, an alkenyl group has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”).In some embodiments, an alkenyl group has 2 to 5 carbon atoms (“C₂₋₅alkenyl”). In some embodiments, an alkenyl group has 2 to 4 carbon atoms(“C₂₋₄ alkenyl”). In some embodiments, an alkenyl group has 2 to 3carbon atoms (“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has2 carbon atoms (“C₂ alkenyl”). The one or more carbon-carbon doublebonds can be internal (such as in 2-butenyl) or terminal (such as in1-butenyl). Examples of C₂₋₄ alkenyl groups include ethenyl (C₂),1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄),butadienyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄ alkenyl groups as well as pentenyl (C₅),pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples ofalkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and thelike. In certain embodiments, each instance of an alkenyl group isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) withone or more substituents. In certain embodiments, the alkenyl group isunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis substituted C₂₋₁₀ alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 20 carbon atoms and one ormore carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds), andoptionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C₂₋₂₀ alkynyl”). In certain embodiments, alkynyl does not comprisedouble bonds. In some embodiments, an alkynyl group has 2 to 10 carbonatoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, analkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In someembodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”).In some embodiments, an alkynyl group has 2 to 5 carbon atoms (“C₂₋₅alkynyl”). In some embodiments, an alkynyl group has 2 to 4 carbon atoms(“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has 2 to 3carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynyl group has2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbon triplebonds can be internal (such as in 2-butynyl) or terminal (such as in1-butynyl). Examples of C₂₋₄ alkynyl groups include, without limitation,ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄),2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groups includethe aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl(C₆), and the like. Additional examples of alkynyl include heptynyl(C₇), octynyl (C₈), and the like. In certain embodiments, each instanceof an alkynyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is substituted C₂₋₁₀ alkynyl.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclyl ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. In certain embodiments, each instance of acarbocyclyl group is independently optionally substituted, e.g.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). In certain embodiments,each instance of a cycloalkyl group is independently unsubstituted (an“unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”)with one or more substituents. In certain embodiments, the cycloalkylgroup is unsubstituted C₃₋₁₀ cycloalkyl. In certain embodiments, thecycloalkyl group is substituted C₃₋₁₀ cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-10 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or a fused, bridged or spiro ring system such as a bicyclic system(“bicyclic heterocyclyl”), and can be saturated or can be partiallyunsaturated. Heterocyclyl bicyclic ring systems can include one or moreheteroatoms in one or both rings. “Heterocyclyl” also includes ringsystems wherein the heterocyclyl ring, as defined above, is fused withone or more carbocyclyl groups wherein the point of attachment is eitheron the carbocyclyl or heterocyclyl ring, or ring systems wherein theheterocyclyl ring, as defined above, is fused with one or more aryl orheteroaryl groups, wherein the point of attachment is on theheterocyclyl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heterocyclylring system. In certain embodiments, each instance of heterocyclyl isindependently optionally substituted, e.g., unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. Incertain embodiments, the heterocyclyl group is substituted 3-10 memberedheterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiorenyl.Exemplary 4-membered heterocyclyl groups containing one heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing one heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, triazinanyl.Exemplary 7-membered heterocyclyl groups containing one heteroatominclude, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl, and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4 n+2 aromatic ring system (e.g., having 6, 10, or 14 πelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₋₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. In certainembodiments, each instance of an aryl group is independently optionallysubstituted, e.g., unsubstituted (an “unsubstituted aryl”) orsubstituted (a “substituted aryl”) with one or more substituents. Incertain embodiments, the aryl group is unsubstituted C₆₋₁₄ aryl. Incertain embodiments, the aryl group is substituted C₆₋₁₄ aryl.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, e.g., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. In certainembodiments, each instance of a heteroaryl group is independentlyoptionally substituted, e.g., unsubstituted (“unsubstituted heteroaryl”)or substituted (“substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group issubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.Exemplary 5,6-bicyclic heteroaryl groups include, without limitation,any one of the following formulae:

In any of the monocyclic or bicyclic heteroaryl groups, the point ofattachment can be any carbon or nitrogen atom, as valency permits.

“Partially unsaturated” refers to a group that includes at least onedouble or triple bond. The term “partially unsaturated” is intended toencompass rings having multiple sites of unsaturation, but is notintended to include aromatic groups (e.g., aryl or heteroaryl groups) asherein defined. Likewise, “saturated” refers to a group that does notcontain a double or triple bond, i.e., contains all single bonds.

In some embodiments, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl groups, as defined herein, are optionallysubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted”or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” carbocyclyl, “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted”, whether preceded by the term “optionally” or not, meansthat at least one hydrogen present on a group (e.g., a carbon ornitrogen atom) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group has a substituent at one ormore substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. The term “substituted” iscontemplated to include substitution with all permissible substituentsof organic compounds, including any of the substituents described hereinthat results in the formation of a stable compound. The presentdisclosure contemplates any and all such combinations in order to arriveat a stable compound. For purposes of this disclosure, heteroatoms suchas nitrogen may have hydrogen substituents and/or any suitablesubstituent as described herein which satisfy the valencies of theheteroatoms and results in the formation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR, —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc); each instance of R^(aa)is, independently, selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(cc))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, or two R^(bb) groups are joined to form a 3-14membered heterocyclyl or 5-14 membered heteroaryl ring, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃+X, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee), —NRCO₂R^(ee),—NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee),OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂,—NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂,—SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —S(═O)R^(ee), —Si(R^(ee))₃,—OSi(R^(ee))₃, —C(═S)N(R^(ff))₂, —C(═O)SR^(ee), —C(═S)SR^(ee),—SC(═S)SR^(ee), —P(═O)₂R^(ee), —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂,—OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 R^(gg) groups, or two geminal R^(dd) substituents canbe joined to form ═O or ═S;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R^(ff) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₄alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₄ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl),—OC(NH)NH₂, —NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl,—SO₂OC₁₋₆ alkyl, —OSO₂Cl₄ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₄ alkyl)₃,—OSi(C₁₋₆ alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆alkyl), —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,C₃₋₁₀ carbocyclyl, C₆-10 aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X is a counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

“Halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro,—Cl), bromine (bromo, —Br), or iodine (iodo, —I).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substitutents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc), and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3rd edition, John Wiley & Sons, 1999, incorporated herein by reference.

Amide nitrogen protecting groups (e.g., —C(═O)R^(aa)) include, but arenot limited to, formamide, acetamide, chloroacetamide,trichloroacetamide, trifluoroacetamide, phenylacetamide,3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide,N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide,o-nitophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide,(N′-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propanamide,3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine,o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

Carbamate nitrogen protecting groups (e.g., —C(═O)OR^(aa)) include, butare not limited to, methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Sulfonamide nitrogen protecting groups (e.g., —S(═O)₂R^(aa)) include,but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxyl)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-naphthylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on a sulfur atom is asulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The present disclosureis not intended to be limited in any manner by the above exemplarylisting of substituents.

“Pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and other animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds describe herein include those derived from suitable inorganicand organic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid, or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, quaternary salts.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (e.g., a male or female of any age group, e.g., apediatric subject (e.g, infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or othernon-human animals, for example, non-human mammals (e.g., primates (e.g.,cynomolgus monkeys, rhesus monkeys); commercially relevant mammals suchas cattle, pigs, horses, sheep, goats, cats, and/or dogs), birds (e.g.,commercially relevant birds such as chickens, ducks, geese, and/orturkeys), rodents (e.g., rats and/or mice), reptiles, amphibians, andfish. In certain embodiments, the non-human animal is a mammal. Thenon-human animal may be a male or female at any stage of development. Anon-human animal may be a transgenic animal.

“Condition,” “disease,” and “disorder” are used interchangeably herein.

“Treat,” “treating” and “treatment” encompasses an action that occurswhile a subject is suffering from a condition which reduces the severityof the condition or retards or slows the progression of the condition(“therapeutic treatment”). “Treat,” “treating” and “treatment” alsoencompasses an action that occurs before a subject begins to suffer fromthe condition and which inhibits or reduces the severity of thecondition (“prophylactic treatment”).

An “effective amount” of a compound refers to an amount sufficient toelicit the desired biological response, e.g., treat the condition. Aswill be appreciated by those of ordinary skill in this art, theeffective amount of a compound described herein may vary depending onsuch factors as the desired biological endpoint, the pharmacokinetics ofthe compound, the condition being treated, the mode of administration,and the age and health of the subject. An effective amount encompassestherapeutic and prophylactic treatment.

A “therapeutically effective amount” of a compound is an amountsufficient to provide a therapeutic benefit in the treatment of acondition or to delay or minimize one or more symptoms associated withthe condition. A therapeutically effective amount of a compound means anamount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms orcauses of the condition, or enhances the therapeutic efficacy of anothertherapeutic agent.

A “prophylactically effective amount” of a compound is an amountsufficient to prevent a condition, or one or more symptoms associatedwith the condition or prevent its recurrence. A prophylacticallyeffective amount of a compound means an amount of a therapeutic agent,alone or in combination with other agents, which provides a prophylacticbenefit in the prevention of the condition. The term “prophylacticallyeffective amount” can encompass an amount that improves overallprophylaxis or enhances the prophylactic efficacy of anotherprophylactic agent.

As used herein, the term “methyltransferase” represents transferaseclass enzymes that are able to transfer a methyl group from a donormolecule to an acceptor molecule, e.g., an amino acid residue of aprotein or a nucleic base of a DNA molecule. Methytransferases typicallyuse a reactive methyl group bound to sulfur in S-adenosyl methionine(SAM) as the methyl donor. In some embodiments, a methyltransferasedescribed herein is a protein methyltransferase. In some embodiments, amethyltransferase described herein is a histone methyltransferase.Histone methyltransferases (HMT) are histone-modifying enzymes,(including histone-lysine N-methyltransferase and histone-arginineN-methyltransferase), that catalyze the transfer of one or more methylgroups to lysine and arginine residues of histone proteins. In certainembodiments, a methyltransferase described herein is a histone-arginineN-methyltransferase.

As generally described above, provided herein are compounds useful asPRMT1 inhibitors. In some embodiments, the present disclosure provides acompound of Formula (I):

or a pharmaceutically acceptable salt thereof,wherein

X is N, Z is NR⁴, and Y is CR⁵; or

X is NR⁴, Z is N, and Y is CR⁵; or

X is CR⁵, Z is NR⁴, and Y is N; or

X is CR⁵, Z is N, and Y is NR⁴;

R² is hydrogen, halo, —CN, —NO₂, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,—OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A),—C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂,—NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂,—NR^(B)C(O)OR^(A), —SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NORA)R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A),—OS(O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂, wherein R²is not —CF₃ or —CHF₂;

R⁶ is hydrogen, halo, —CN, —NO₂, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted aryl,optionally substituted heterocyclyl, optionally substituted heteroaryl,—OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A),—C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂,—NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂,—NR^(B)C(O)OR^(A), —SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NORA)R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A),—OS(O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂, wherein R⁶is not —CF₃ or —CHF₂;

each R^(A) is independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, and a sulfur protecting group when attachedto a sulfur atom;

each R^(B) is independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl, andoptionally substituted heteroaryl, and a nitrogen protecting group, ortwo R^(B) groups are taken together with their intervening atoms to forman optionally substituted heterocyclic ring;

R³ is hydrogen, C₁₋₄ alkyl, or C₃₋₄ cycloalkyl;

R⁴ is hydrogen, optionally substituted C₁₋₆ alkyl, optionallysubstituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl,optionally substituted C₃₋₇ cycloalkyl, optionally substituted 4- to7-membered heterocyclyl; or optionally substituted C₁₋₄ alkyl-Cy;

Cy is optionally substituted C₃₋₇ cycloalkyl, optionally substituted 4-to 7-membered heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl;

R⁵ is hydrogen, halo, —CN, optionally substituted C₁₋₄ alkyl, oroptionally substituted C₃₋₄ cycloalkyl;

R^(x) is optionally substituted C₁₋₄ alkyl or optionally substitutedC₃₋₄ cycloalkyl;

R^(y) is —NR^(B)C(O)R^(A), —NR^(B)SO₂R^(A), or—(CR^(z)R^(z))_(n)C(O)N(R^(B))₂;

each R^(z) is independently hydrogen or fluoro; and

n is 0, 1, 2, 3, or 4.

In certain embodiments, a provided compound is of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein R^(y), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (II-a):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R³,R⁴, R⁵, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (II-b):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R³,R⁴, R⁵, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (II-c):

or a pharmaceutically acceptable salt thereof, wherein R^(B), R³, R⁴,R⁵, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein R^(y), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (III-a):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R²,R³, R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (III-b):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R²,R³, R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (III-c):

or a pharmaceutically acceptable salt thereof, wherein R^(B), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein R^(y), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (IV-a):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R²,R³, R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (IV-b):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R²,R³, R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (IV-c):

or a pharmaceutically acceptable salt thereof, wherein R^(B), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein R^(y), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (V-a):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R²,R³, R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (V-b):

or a pharmaceutically acceptable salt thereof, wherein R^(A), R^(B), R²,R³, R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, a provided compound is of Formula (V-c):

or a pharmaceutically acceptable salt thereof, wherein R^(B), R², R³,R⁴, R⁵, R⁶, and R^(x) are as described herein.

In certain embodiments, R⁶ is hydrogen and R² is not hydrogen. Incertain embodiments, R⁶ is not hydrogen and R² is hydrogen. In certainembodiments, R⁶ and R² are hydrogen. In certain embodiments, R⁶ and R²are not hydrogen.

As defined generally above, R^(y) is —NR^(B)C(O)R^(A), —NR^(B)SO₂R^(A),or —(CR^(z)R^(z))_(n)C(O)N(R^(B))₂. In certain embodiments, R^(y) is—NR^(B)C(O)R^(A). In certain embodiments, R^(y) is —NHC(O)R^(A). Incertain embodiments, R^(y) is —NR^(B)C(O)R^(A) or —NHC(O)R^(A), whereinR^(A) is optionally substituted alkyl. In certain embodiments, R^(y) is—NR^(B)C(O)R^(A) or —NHC(O)R^(A), wherein R^(A) is alkoxyalkyl. Incertain embodiments, R^(y) is —NR^(B)C(O)R^(A) or —NHC(O)R^(A), whereinR^(A) is optionally substituted alkenyl. In certain embodiments, R^(y)is —NR^(B)C(O)R^(A) or —NHC(O)R^(A), wherein R^(A) is unsubstituted C₁₋₆alkyl. In certain embodiments, R^(y) is —NR^(B)SO₂R^(A) In certainembodiments, R^(y) is —NHSO₂R^(A). In certain embodiments, R^(y) is—NR^(B)SO₂R^(A) or —NHSO₂R^(A), wherein R^(A) is optionally substitutedalkyl. In certain embodiments, R^(y) is —NR^(B)SO₂R^(A) or —NHSO₂R^(A),wherein R^(A) is alkoxyalkyl. In certain embodiments, R^(y) is—NR^(B)SO₂R^(A) or —NHSO₂R^(A), wherein R^(A) is optionally substitutedalkenyl. In certain embodiments, R^(y) is —NR^(B)SO₂R^(A) or—NHSO₂R^(A), wherein R^(A) is unsubstituted C₁₋₆ alkyl. In certainembodiments, R^(y) is —(CR^(z)R^(z))_(n)C(O)N(R^(B))₂. In certainembodiments, R^(y) is —CH₂C(O)N(R^(B))₂. In certain embodiments, R^(y)is —(CR^(z)R^(z))_(n)C(O)N(R^(B))₂ or —CH₂C(O)N(R^(B))₂, wherein R^(A)is optionally substituted alkyl. In certain embodiments, R^(y) is—(CR^(z)R^(z))_(n)C(O)N(R^(B))₂ or —CH₂C(O)N(R^(B))₂, wherein R^(A) isalkoxyalkyl. In certain embodiments, R^(y) is—(CR^(z)R^(z))_(n)C(O)N(R^(B))₂ or —CH₂C(O)N(R^(B))₂, wherein R^(A) isoptionally substituted alkenyl. In certain embodiments, R^(y) is—(CR^(z)R^(z))_(n)C(O)N(R^(B))₂ or —CH₂C(O)N(R^(B))₂, wherein R^(A) isunsubstituted C₁₋₆ alkyl. In certain embodiments, each R^(z) ishydrogen. In certain embodiments, n is 0. In certain embodiments, nis 1. In certain embodiments, n is 2. In certain embodiments, n is 3. Incertain embodiments, n is 4. In some embodiments, R^(A) is optionallysubstituted C₁₋₆ alkyl. In some embodiments, R^(A) is C₁₋₄ alkyl. Insome embodiments, R^(A) is C₂₋₄ alkenyl. In some embodiments, R^(A) isC₂₋₄ alkynyl. In some embodiments, R^(B) is optionally substituted C₁₋₆alkyl. In some embodiments, R^(B) is C₁₋₄ alkyl. In some embodiments,R^(B) is C₂₋₄ alkenyl. In some embodiments, R^(B) is C₂₋₄ alkynyl.

As defined generally above, R² is hydrogen, halo, —CN, —NO₂, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(O)OR^(A), —C(O)SR^(A), —C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂,—OC(O)R^(A), —OC(O)N(R^(B))₂, —NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂,—NR^(B)C(O)N(R^(B))N(R^(B))₂, —NR^(B)C(O)OR^(A), —SC(O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NORA)R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A), —OS(O)₂R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In some embodiments, R² is not —CF₃or —CHF₂. In certain embodiments, R² is hydrogen. In some embodiments,R² is not hydrogen. In some embodiments, R² is halo. In certainembodiments, R² is fluoro. In certain embodiments, R² is chloro. In someembodiments, R² is optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, or optionally substitutedcarbocyclyl. In certain embodiments, R² is optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆alkynyl, or optionally substituted C₃₋₆ carbocyclyl. In certainembodiments, R² is optionally substituted C₁₋₆ alkyl. In certainembodiments, R² is substituted C₁₋₆ alkyl. In certain embodiments, R² is—C₁₋₆alkyl-carbocyclyl. In certain embodiments, R² is —CH₂-cyclopropylor —CH₂-cyclobutyl. In certain embodiments, R² is unsubstituted C₁₋₆alkyl. In certain embodiments, R² is methyl, ethyl, propyl, butyl, orpentyl. In certain embodiments, R² is isopropyl, isobutyl, or isoamyl.In certain embodiments, R² is isobutyl. In some embodiments, R² is —CN.In some embodiments, R² is optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl, oroptionally substituted heteroaryl. In some embodiments, R² is —OR^(A),—N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A),—C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂,—NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂,—NR^(B)C(O)OR^(A), —SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NORA)R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A),—OS(O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In certainembodiments, R² is —N(R^(B))₂. In certain embodiments, R² is —NHR^(B).In certain embodiments, R² is —NH₂. In certain embodiments, R² is—OR^(A). In certain embodiments, R² is —OH. In certain embodiments, R²is —OR^(A), wherein R^(A) is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted carbocyclyl. In certain embodiments, R² is —O— isobutylenyl.In certain embodiments, R² is —OR^(A), wherein R^(A) is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, R² is —OR^(A), whereinR^(A) is unsubstituted C₁₋₆ alkyl. In certain embodiments, R² is—O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certainembodiments, R² is —OCH(CH₂CH₃)₂. In certain embodiments, R² is—OCH(OH)CH(CH₃)₂. In certain embodiments, R² is —OR^(A), wherein R^(A)is substituted C₁₋₆ alkyl. In certain embodiments, R² is—O—C₁₋₆alkyl-O—C₁₋₆alkyl. In certain embodiments, R² is —OCH₂CH₂OCH₃,—OCH₂CH₂CH₂OCH₃, —OCH₂CH₂OH, or —OCH₂CH₂OCH₂CH₂CH₃. In certainembodiments, R² is —OCH₂CF₃ or —OCH₂CH₂CH₂CF₃. In certain embodiments,R² is —OCH(CH₃)₂, —OCH₂CH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₂CH(CH₃)₂,—OCH₂CH₂CH₂OCH₃, or —OCH₂CH₂OCH₃. In certain embodiments, R² is—O—C₁₋₆alkyl-carbocyclyl. In certain embodiments, R² is—O—CH₂-cyclobutyl or —O—CH₂-cyclopropyl. In certain embodiments, R² is—O—CH₂-cyclopentyl or —O—CH₂CH₂-cyclohexyl. In certain embodiments, R²is —O—C₁₋₆alkyl-heterocyclyl. In certain embodiments, R² is—O—CH₂-tetrahydropyranyl or —O—CH₂-oxetanyl. In certain embodiments, R²is —O—C₁₋₆alkyl-aryl. In certain embodiments, R² is —O-benzyl or—OCH₂CH₂Ph. In certain embodiments, R² is —O—C₁₋₆alkyl-heteroaryl. Incertain embodiments, R² is —OR^(A), wherein R^(A) is optionallysubstituted heterocyclyl. In certain embodiments, R² is—O-tetrahydropyranyl or —O-oxetanyl. In certain embodiments, R² is—OR^(A), wherein R^(A) is optionally substituted aryl. In certainembodiments, R² is —O-phenyl. In certain embodiments, R is —OR^(A),wherein R^(A) is optionally substituted heteroaryl. In certainembodiments, R² is —O— pyridyl. In certain embodiments, R² is—O-2-pyridyl. In certain embodiments, R² is —O-3-pyridyl. In certainembodiments, R² is —O-4-pyridyl. In certain embodiments, R² is—O-pyrimidinyl.

As defined generally above, R⁶ is hydrogen, halo, —CN, —NO₂, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted aryl, optionally substituted heterocyclyl, optionallysubstituted heteroaryl, —OR^(A), —N(R^(B))₂, —SR^(A), —C(═O)R^(A),—C(O)OR^(A), —C(O)SR^(A), —C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂,—OC(O)R^(A), —OC(O)N(R^(B))₂, —NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂,—NR^(B)C(O)N(R^(B))N(R^(B))₂, —NR^(B)C(O)OR^(A), —SC(O)R^(A),—C(═NR^(B))R^(A), —C(═NNR^(B))R^(A), —C(═NORA)R^(A),—C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B), —C(═S)R^(A),—C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A), —OS(O)₂R^(A), —SO₂R^(A),—NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In certain embodiments, R⁶ ishydrogen. In some embodiments, R⁶ is not hydrogen. In some embodiments,R⁶ is halo. In certain embodiments, R⁶ is fluoro. In certainembodiments, R⁶ is chloro. In some embodiments, R⁶ is optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted carbocyclyl. In certainembodiments, R⁶ is optionally substituted C₁₋₆ alkyl, optionallysubstituted C₂₋₆ alkenyl, optionally substituted C₂₋₆ alkynyl, oroptionally substituted C₃₋₆ carbocyclyl. In certain embodiments, R⁶ isoptionally substituted C₁₋₆ alkyl. In certain embodiments, R⁶ issubstituted C₁₋₆ alkyl. In certain embodiments, R⁶ is—C₁₋₆alkyl-carbocyclyl. In certain embodiments, R⁶ is —CH₂-cyclopropylor —CH₂-cyclobutyl. In certain embodiments, R⁶ is unsubstituted C₁₋₆alkyl. In certain embodiments, R⁶ is methyl, ethyl, propyl, butyl, orpentyl. In certain embodiments, R⁶ is isopropyl, isobutyl, or isoamyl.In certain embodiments, R⁶ is isobutyl. In some embodiments, R⁶ is —CN.In some embodiments, R⁶ is optionally substituted carbocyclyl,optionally substituted aryl, optionally substituted heterocyclyl, oroptionally substituted heteroaryl. In some embodiments, R⁶ is —OR^(A),—N(R^(B))₂, —SR^(A), —C(═O)R^(A), —C(O)OR^(A), —C(O)SR^(A),—C(O)N(R^(B))₂, —C(O)N(R^(B))N(R^(B))₂, —OC(O)R^(A), —OC(O)N(R^(B))₂,—NR^(B)C(O)R^(A), —NR^(B)C(O)N(R^(B))₂, —NR^(B)C(O)N(R^(B))N(R^(B))₂,—NR^(B)C(O)OR^(A), —SC(O)R^(A), —C(═NR^(B))R^(A), —C(═NNR^(B))R^(A),—C(═NORA)R^(A), —C(═NR^(B))N(R^(B))₂, —NR^(B)C(═NR^(B))R^(B),—C(═S)R^(A), —C(═S)N(R^(B))₂, —NR^(B)C(═S)R^(A), —S(O)R^(A),—OS(O)₂R^(A), —SO₂R^(A), —NR^(B)SO₂R^(A), or —SO₂N(R^(B))₂. In certainembodiments, R⁶ is —N(R^(B))₂. In certain embodiments, R⁶ is —NHR^(B).In certain embodiments, R⁶ is —NH₂. In certain embodiments, R⁶ is—OR^(A). In certain embodiments, R⁶ is —OH. In certain embodiments, R⁶is —OR^(A), wherein R^(A) is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted carbocyclyl. In certain embodiments, R⁶ is —O-isobutylenyl.In certain embodiments, R⁶ is —OR^(A), wherein R^(A) is optionallysubstituted C₁₋₆ alkyl. In certain embodiments, R⁶ is —OR^(A), whereinR^(A) is unsubstituted C₁₋₆ alkyl. In certain embodiments, R⁶ is—O-propyl, —O-isopropyl, —O-isobutyl, or —O-isoamyl. In certainembodiments, R⁶ is —OCH(CH₂CH₃)₂. In certain embodiments, R⁶ is—OCH(OH)CH(CH₃)₂. In certain embodiments, R⁶ is —OR^(A), wherein R^(A)is substituted C₁₋₆ alkyl. In certain embodiments, R⁶ is—O—C₁₋₆alkyl-O—C₁₋₆alkyl. In certain embodiments, R⁶ is —OCH₂CH₂OCH₃,—OCH₂CH₂CH₂OCH₃, —OCH₂CH₂OH, or —OCH₂CH₂OCH₂CH₂CH₃. In certainembodiments, R⁶ is —OCH₂CF₃ or —OCH₂CH₂CH₂CF₃. In certain embodiments,R⁶ is —O—C₁₋₆alkyl-carbocyclyl. In certain embodiments, R⁶ is—O—CH₂-cyclobutyl or —O—CH₂-cyclopropyl. In certain embodiments, R⁶ is—O—CH₂-cyclopentyl or —O—CH₂CH₂-cyclohexyl. In certain embodiments, R⁶is —O—C₁₋₆alkyl-heterocyclyl. In certain embodiments, R⁶ is—O—CH₂-tetrahydropyranyl or —O—CH₂-oxetanyl. In certain embodiments, R⁶is —O—C₁₋₆alkyl-aryl. In certain embodiments, R⁶ is —O-benzyl or—OCH₂CH₂Ph. In certain embodiments, R⁶ is —O—C₁₋₆alkyl-heteroaryl. Incertain embodiments, R⁶ is —OR^(A), wherein R^(A) is optionallysubstituted heterocyclyl. In certain embodiments, R⁶ is—O-tetrahydropyranyl or —O— oxetanyl. In certain embodiments, R⁶ is—OR^(A), wherein R^(A) is optionally substituted aryl. In certainembodiments, R⁶ is —O-phenyl. In certain embodiments, R⁶ is —OR^(A),wherein R^(A) is optionally substituted heteroaryl. In certainembodiments, R⁶ is —O-pyridyl. In certain embodiments, R⁶ is—O-2-pyridyl. In certain embodiments, R⁶ is —O-3-pyridyl. In certainembodiments, R⁶ is —O-4-pyridyl. In certain embodiments, R⁶ is—O-pyrimidinyl.

As defined generally above, R³ is hydrogen, C₁₋₄ alkyl, or C₃₋₄cycloalkyl. In certain embodiments, R³ is hydrogen. In certainembodiments, R³ is C₁₋₄ alkyl. In certain embodiments, R³ is methyl. Incertain embodiments, R³ is ethyl. In certain embodiments, R³ is propylor butyl. In certain embodiments, R³ is cyclopropyl. In certainembodiment, R³ is cyclobutyl.

As defined generally above, R⁴ is hydrogen, optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆ alkenyl, optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₇ cycloalkyl, optionally substituted4- to 7-membered heterocyclyl; or optionally substituted C₁₋₄ alkyl-Cy,wherein Cy is optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,or optionally substituted heteroaryl. In certain embodiments, R⁴ ishydrogen. In certain embodiments, R⁴ is optionally substituted C₁₋₆alkyl. In certain embodiments, R⁴ is unsubstituted C₁₋₆ alkyl. Incertain embodiments, R⁴ is methyl, ethyl, or isopropyl. In certainembodiments, R⁴ is substituted C₁₋₆ alkyl. In certain embodiments, R⁴ ismethoxyethyl. In certain embodiments, R⁴ is hydroxyethyl orpropane-1,2-diol. In certain embodiments, R⁴ is optionally substitutedC₃₋₇ cycloalkyl. In certain embodiments, R⁴ is unsubstituted C₃₋₇cycloalkyl. In certain embodiments, R⁴ is cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. In certain embodiments, R⁴ is optionallysubstituted 4- to 7-membered heterocyclyl. In certain embodiments, R⁴ isoptionally substituted 4- to 7-membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, and sulfur. Incertain embodiments, R⁴ is oxetane, tetrahydrofuran, or tetrahydropyran.In certain embodiments, R⁴ is optionally substituted C₁₋₄ alkyl-Cy,wherein Cy is optionally substituted C₃₋₇ cycloalkyl, optionallysubstituted 4- to 7-membered heterocyclyl, optionally substituted aryl,or optionally substituted heteroaryl. In some embodiments, Cy isoptionally substituted C₃₋₇ cycloalkyl. In some embodiments, Cy isoptionally substituted 4- to 7-membered heterocyclyl having 1-2heteroatoms independently selected from nitrogen, oxygen, and sulfur. Insome embodiments, Cy is oxetane, tetrahydrofuran, or tetrahydropyran. Insome embodiments, Cy is optionally substituted aryl. In someembodiments, Cy is optionally substituted phenyl. In some embodiments,Cy is unsubstituted phenyl. In some embodiments, Cy is optionallysubstituted heteroaryl having 1-3 heteroatoms independently selectedfrom nitrogen, oxygen, and sulfur. In some embodiments, Cy is optionallysubstituted 5- to 6-membered heteroaryl having 1-3 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. In someembodiments, Cy is pyridyl. In some embodiments, R⁴ is

In some embodiments, R⁴ is

In some embodiments, R⁴ is

As defined generally above, R⁵ is hydrogen, halo, —CN, optionallysubstituted C₁₋₄ alkyl, or optionally substituted C₃₋₄ cycloalkyl. Incertain embodiments, R⁵ is hydrogen. In certain embodiments, R⁵ is halo.In certain embodiments, R⁵ is chloro. In certain embodiments, R⁵ isfluoro. In certain embodiments, R⁵ is —CN. In certain embodiments, R⁵ isoptionally substituted C₁₋₄ alkyl. In certain embodiments, R⁵ isunsubstituted C₁₋₄ alkyl. In certain embodiments, R⁵ is methyl. Incertain embodiments, R⁵ is ethyl. In certain embodiments, R⁵ is propylor butyl. In certain embodiments, R⁵ is C₁₋₄ alkyl substituted with oneor more fluoro groups. In certain embodiments, R⁵ is —CF₃. In certainembodiments, R⁵ is —CHF₂. In certain embodiments, R⁵ is optionallysubstituted C₃₋₄ cycloalkyl. In certain embodiments, R⁵ is cyclopropylor cyclobutyl.

As defined generally above, R^(x) is optionally substituted C₁₋₄ alkylor optionally substituted C₃₋₄ cycloalkyl. In certain embodiments, R^(x)is optionally substituted C₁₋₄ alkyl. In certain embodiments, R^(x) isunsubstituted C₁₋₄ alkyl. In certain embodiments, R^(x) is methyl. Incertain embodiments, R^(x) is ethyl. In certain embodiments, R^(x) isisopropyl. In certain embodiments, R^(x) is propyl or butyl. In certainembodiments, R^(x) is substituted C₁₋₄ alkyl. In certain embodiments,R^(x) is C₁₋₄ alkyl substituted with hydroxyl or alkoxy. In certainembodiments, R^(x) is hydroxyethyl or methoxyethyl. In certainembodiments, R^(x) is optionally substituted C₃₋₄ cycloalkyl. In certainembodiments, R^(x) is unsubstituted C₃₋₄ cycloalkyl. In certainembodiments, R^(x) is cyclopropyl. In certain embodiments, R^(x) iscyclobutyl.

As defined generally above, each R^(A) is independently selected fromthe group consisting of hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, and a sulfurprotecting group when attached to a sulfur atom. In some embodiments,R^(A) is hydrogen. In some embodiments, R^(A) is optionally substitutedalkyl. In some embodiments, R^(A) is optionally substituted alkylsubstituted with a Cy group to form optionally substituted alkyl-Cy,wherein Cy is described herein. In some embodiments, R^(A) is optionallysubstituted alkenyl or optionally substituted alkynyl. In someembodiments, R^(A) is optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, or optionallysubstituted heteroaryl. In some embodiments, R^(A) is an oxygenprotecting group when attached to an oxygen atom. In some embodiments,R^(A) is not an oxygen protecting group. In some embodiments, R^(A) issulfur protecting group when attached to an sulfur atom. In someembodiments, R^(A) is not a sulfur protecting group.

As defined generally above, each R^(B) is independently selected fromthe group consisting of hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, and optionally substituted heteroaryl, anda nitrogen protecting group, or two R^(B) groups are taken together withtheir intervening atoms to form an optionally substituted heterocyclicring. In some embodiments, R^(B) is hydrogen. In some embodiments, R^(B)is optionally substituted alkyl. In some embodiments, R^(B) isoptionally alkyl substituted with a Cy group to form optionallysubstituted alkyl-Cy, wherein Cy is described herein. In someembodiments, R^(B) is optionally substituted alkenyl or optionallysubstituted alkynyl. In some embodiments, R^(B) is optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In someembodiments, R^(B) is a nitrogen protecting group. In some embodiments,R^(B) is not a nitrogen protecting group. In some embodiments, two R^(B)groups are taken together with their intervening atoms to form anoptionally substituted heterocyclic ring.

In certain embodiments, a provided compound is a compound listed inTable 1, or a pharmaceutically acceptable salt thereof.

TABLE 1 Exemplary Compounds LC- MS m/z Cmpd (M + No Structure H) 1

328.3 2

314.0 3

342.3 4

5

6

7

8

330.2 9

10

11

12

13

14

15

398.1

In certain embodiments, a provided compound inhibits PRMT1. In certainembodiments, a provided compound inhibits wild-type PRMT1. In certainembodiments, a provided compound inhibits a mutant PRMT1. In certainembodiments, a provided compound inhibits PRMT1, e.g., as measured in anassay described herein. In certain embodiments, the PRMT1 is from ahuman. In certain embodiments, a provided compound inhibits PRMT1 at anIC₅₀ less than or equal to 10 μM. In certain embodiments, a providedcompound inhibits PRMT1 at an IC₅₀ less than or equal to 1 μM. Incertain embodiments, a provided compound inhibits PRMT1 at an IC₅₀ lessthan or equal to 0.1 μM. In certain embodiments, a provided compoundinhibits PRMT1 at an IC50 less than or equal to 0.01 μM. In certainembodiments, a provided compound inhibits PRMT1 in a cell at an EC₃₀less than or equal to 10 μM. In certain embodiments, a provided compoundinhibits PRMT1 in a cell at an EC₃₀ less than or equal to 12 μM. Incertain embodiments, a provided compound inhibits PRMT1 in a cell at anEC₃₀ less than or equal to 3 μM. In certain embodiments, a providedcompound inhibits PRMT1 in a cell at an EC₃₀ less than or equal to 1 μM.In certain embodiments, a provided compound inhibits PRMT1 in a cell atan EC₃₀ less than or equal to 0.1 μM. In certain embodiments, a providedcompound inhibits cell proliferation at an EC₅₀ less than or equal to 10μM. In certain embodiments, a provided compound inhibits cellproliferation at an EC₅₀ less than or equal to 1 μM. In certainembodiments, a provided compound inhibits cell proliferation at an EC₅₀less than or equal to 0.1 μM. In some embodiments, a provided compoundis selective for PRMT1 over other methyltransferases. In certainembodiments, a provided compound is at least about 10-fold selective, atleast about 20-fold selective, at least about 30-fold selective, atleast about 40-fold selective, at least about 50-fold selective, atleast about 60-fold selective, at least about 70-fold selective, atleast about 80-fold selective, at least about 90-fold selective, or atleast about 100-fold selective for PRMT1 relative to one or more othermethyltransferases (e.g., all other methyltransferases).

It will be understood by one of ordinary skill in the art that the PRMT1can be wild-type PRMT1, or any mutant or variant of PRMT1.

The present disclosure provides pharmaceutical compositions comprising acompound described herein, e.g., a compound of Formula (I), or apharmaceutically acceptable salt thereof, as described herein, andoptionally a pharmaceutically acceptable excipient. It will beunderstood by one of ordinary skill in the art that the compoundsdescribed herein, or salts thereof, may be present in various forms,such as amorphous, hydrates, solvates, or polymorphs. In certainembodiments, a provided composition comprises two or more compoundsdescribed herein. In certain embodiments, a compound described herein,or a pharmaceutically acceptable salt thereof, is provided in aneffective amount in the pharmaceutical composition. In certainembodiments, the effective amount is a therapeutically effective amount.In certain embodiments, the effective amount is an amount effective forinhibiting PRMT1. In certain embodiments, the effective amount is anamount effective for treating a PRMT1-mediated disorder. In certainembodiments, the effective amount is a prophylactically effectiveamount. In certain embodiments, the effective amount is an amounteffective to prevent a PRMT1-mediated disorder.

Pharmaceutically acceptable excipients include any and all solvents,diluents, or other liquid vehicles, dispersions, suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants, and the like, assuited to the particular dosage form desired. General considerations informulation and/or manufacture of pharmaceutical compositions agents canbe found, for example, in Remington's Pharmaceutical Sciences, SixteenthEdition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), andRemington: The Science and Practice of Pharmacy, 21st Edition(Lippincott Williams & Wilkins, 2005).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include the steps of bringing a compound described herein (the“active ingredient”) into association with a carrier and/or one or moreother accessory ingredients, and then, if necessary and/or desirable,shaping and/or packaging the product into a desired single- ormulti-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition of the present disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100% (w/w) active ingredient.

In some embodiments, a pharmaceutical composition described herein issterilized.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calciumphosphate, dicalcium phosphate, calcium sulfate, calcium hydrogenphosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch,corn starch, tapioca starch, sodium starch glycolate, clays, alginicacid, guar gum, citrus pulp, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, calcium carbonate,silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)(crospovidone), sodium carboxymethyl starch (sodium starch glycolate),carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),microcrystalline starch, water insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate,quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include naturalemulsifiers (e.g., acacia, agar, alginic acid, sodium alginate,tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk,casein, wool fat, cholesterol, wax, and lecithin), colloidal clays(e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminumsilicate)), long chain amino acid derivatives, high molecular weightalcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.,carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylenesorbitan monolaurate (Tween 20), polyoxyethylene sorbitan (Tween 60),polyoxyethylene sorbitan monooleate (Tween 80), sorbitan monopalmitate(Span 40), sorbitan monostearate (Span 60], sorbitan tristearate (Span65), glyceryl monooleate, sorbitan monooleate (Span 80)),polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj 45),polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g., Cremophor™),polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij 30)),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimoniumbromide, cetylpyridinium chloride, benzalkonium chloride, docusatesodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starchpaste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin,molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums(e.g., acacia, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapol husks, carboxymethylcellulose,methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate(Veegum), and larch arabogalactan), alginates, polyethylene oxide,polyethylene glycol, inorganic calcium salts, silicic acid,polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene,monothioglycerol, potassium metabisulfite, propionic acid, propylgallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, andsodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid(EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodiumedetate, trisodium edetate, calcium disodium edetate, dipotassiumedetate, and the like), citric acid and salts and hydrates thereof(e.g., citric acid monohydrate), fumaric acid and salts and hydratesthereof, malic acid and salts and hydrates thereof, phosphoric acid andsalts and hydrates thereof, and tartaric acid and salts and hydratesthereof. Exemplary antimicrobial preservatives include benzalkoniumchloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide,cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea,phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate,propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methylparaben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoicacid, potassium benzoate, potassium sorbate, sodium benzoate, sodiumpropionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol,phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate,and phenylethyl alcohol. Exemplary acidic preservatives include vitaminA, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid,dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroximemesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus,Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, andEuxyl. In certain embodiments, the preservative is an anti-oxidant. Inother embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetatebuffer solutions, phosphate buffer solutions, ammonium chloride, calciumcarbonate, calcium chloride, calcium citrate, calcium glubionate,calcium gluceptate, calcium gluconate, D-gluconic acid, calciumglycerophosphate, calcium lactate, propanoic acid, calcium levulinate,pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasiccalcium phosphate, calcium hydroxide phosphate, potassium acetate,potassium chloride, potassium gluconate, potassium mixtures, dibasicpotassium phosphate, monobasic potassium phosphate, potassium phosphatemixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodiumcitrate, sodium lactate, dibasic sodium phosphate, monobasic sodiumphosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide,aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline,Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calciumstearate, stearic acid, silica, talc, malt, glyceryl behanate,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate,sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu,bergamot, black current seed, borage, cade, camomile, canola, caraway,carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee,corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed,geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate,jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, andwheat germ oils. Exemplary synthetic oils include, but are not limitedto, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixturesthereof.

Liquid dosage forms for oral and parenteral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredients,the liquid dosage forms may comprise inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and perfuming agents. Incertain embodiments for parenteral administration, the compoundsdescribed herein are mixed with solubilizing agents such as Cremophor™,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the compounds describedherein with suitable non-irritating excipients or carriers such as cocoabutter, polyethylene glycol or a suppository wax which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may comprise buffering agents.

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type can be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

The active ingredient can be in micro-encapsulated form with one or moreexcipients as noted above. The solid dosage forms of tablets, dragees,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active ingredient can be admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms maycomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets, and pills, the dosage forms may comprise bufferingagents. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a providedcompound may include ointments, pastes, creams, lotions, gels, powders,solutions, sprays, inhalants and/or patches. Generally, the activeingredient is admixed under sterile conditions with a pharmaceuticallyacceptable carrier and/or any desired preservatives and/or buffers ascan be required. Additionally, the present disclosure encompasses theuse of transdermal patches, which often have the added advantage ofproviding controlled delivery of an active ingredient to the body. Suchdosage forms can be prepared, for example, by dissolving and/ordispensing the active ingredient in the proper medium. Alternatively oradditionally, the rate can be controlled by either providing a ratecontrolling membrane and/or by dispersing the active ingredient in apolymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient can be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation may comprise dry particles whichcomprise the active ingredient and which have a diameter in the rangefrom about 0.5 to about 7 nanometers or from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder and/or using a self propelling solvent/powder dispensingcontainer such as a device comprising the active ingredient dissolvedand/or suspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions mayinclude a solid fine powder diluent such as sugar and are convenientlyprovided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic and/or solid anionic surfactant and/or a solid diluent(which may have a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide the active ingredient in the form of droplets of a solutionand/or suspension. Such formulations can be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising the active ingredient, and mayconveniently be administered using any nebulization and/or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, and/or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration may have an average diameter inthe range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 to 500 micrometers. Such a formulation is administered byrapid inhalation through the nasal passage from a container of thepowder held close to the nares.

Formulations for nasal administration may, for example, comprise fromabout as little as 0.1% (w/w) and as much as 100% (w/w) of the activeingredient, and may comprise one or more of the additional ingredientsdescribed herein. A provided pharmaceutical composition can be prepared,packaged, and/or sold in a formulation for buccal administration. Suchformulations may, for example, be in the form of tablets and/or lozengesmade using conventional methods, and may contain, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable and/or degradable composition and, optionally, one or moreof the additional ingredients described herein. Alternately,formulations for buccal administration may comprise a powder and/or anaerosolized and/or atomized solution and/or suspension comprising theactive ingredient. Such powdered, aerosolized, and/or aerosolizedformulations, when dispersed, may have an average particle and/ordroplet size in the range from about 0.1 to about 200 nanometers, andmay further comprise one or more of the additional ingredients describedherein.

A provided pharmaceutical composition can be prepared, packaged, and/orsold in a formulation for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid carrier. Such drops may further comprisebuffering agents, salts, and/or one or more other of the additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis disclosure.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with ordinary experimentation.

Compounds provided herein are typically formulated in dosage unit formfor ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of provided compositionswill be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective dose level forany particular subject or organism will depend upon a variety of factorsincluding the disease, disorder, or condition being treated and theseverity of the disorder; the activity of the specific active ingredientemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific activeingredient employed; the duration of the treatment; drugs used incombination or coincidental with the specific active ingredientemployed; and like factors well known in the medical arts.

The compounds and compositions provided herein can be administered byany route, including enteral (e.g., oral), parenteral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),and/or the condition of the subject (e.g., whether the subject is ableto tolerate oral administration).

The exact amount of a compound required to achieve an effective amountwill vary from subject to subject, depending, for example, on species,age, and general condition of a subject, severity of the side effects ordisorder, identity of the particular compound(s), mode ofadministration, and the like. The desired dosage can be delivered threetimes a day, two times a day, once a day, every other day, every thirdday, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage can be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

In certain embodiments, an effective amount of a compound foradministration one or more times a day to a 70 kg adult human maycomprise about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg,about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosageform.

In certain embodiments, a compound described herein may be administeredat dosage levels sufficient to deliver from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about mg/kg, from about 0.1 mg/kgto about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, orfrom about 1 mg/kg to about 25 mg/kg, of subject body weight per day,one or more times a day, to obtain the desired therapeutic effect.

In some embodiments, a compound described herein is administered one ormore times per day, for multiple days. In some embodiments, the dosingregimen is continued for days, weeks, months, or years.

It will be appreciated that dose ranges as described herein provideguidance for the administration of provided pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

It will be also appreciated that a compound or composition, as describedherein, can be administered in combination with one or more additionaltherapeutically active agents. In certain embodiments, a compound orcomposition provided herein is administered in combination with one ormore additional therapeutically active agents that improve itsbioavailability, reduce and/or modify its metabolism, inhibit itsexcretion, and/or modify its distribution within the body. It will alsobe appreciated that the therapy employed may achieve a desired effectfor the same disorder, and/or it may achieve different effects.

The compound or composition can be administered concurrently with, priorto, or subsequent to, one or more additional therapeutically activeagents. In certain embodiments, the additional therapeutically activeagent is a compound of Formula (I). In certain embodiments, theadditional therapeutically active agent is not a compound of Formula(I). In general, each agent will be administered at a dose and/or on atime schedule determined for that agent. In will further be appreciatedthat the additional therapeutically active agent utilized in thiscombination can be administered together in a single composition oradministered separately in different compositions. The particularcombination to employ in a regimen will take into account compatibilityof a provided compound with the additional therapeutically active agentand/or the desired therapeutic effect to be achieved. In general, it isexpected that additional therapeutically active agents utilized incombination be utilized at levels that do not exceed the levels at whichthey are utilized individually. In some embodiments, the levels utilizedin combination will be lower than those utilized individually.

Exemplary additional therapeutically active agents include, but are notlimited to, small organic molecules such as drug compounds (e.g.,compounds approved by the U.S. Food and Drug Administration as providedin the Code of Federal Regulations (CFR)), peptides, proteins,carbohydrates, monosaccharides, oligosaccharides, polysaccharides,nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides orproteins, small molecules linked to proteins, glycoproteins, steroids,nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides,antisense oligonucleotides, lipids, hormones, vitamins, and cells. Incertain embodiments, an additional therapeutically active agent isprednisolone, dexamethasone, doxorubicin, vincristine, mafosfamide,cisplatin, carboplatin, Ara-C, rituximab, azacitadine, panobinostat,vorinostat, everolimus, rapamycin, ATRA (all-trans retinoic acid),daunorubicin, decitabine, Vidaza, mitoxantrone, or IBET-151.

Also encompassed by the present disclosure are kits (e.g.,pharmaceutical packs). The kits provided may comprise a providedpharmaceutical composition or compound and a container (e.g., a vial,ampule, bottle, syringe, and/or dispenser package, or other suitablecontainer). In some embodiments, provided kits may optionally furtherinclude a second container comprising a pharmaceutical excipient fordilution or suspension of a provided pharmaceutical composition orcompound. In some embodiments, a provided pharmaceutical composition orcompound provided in the container and the second container are combinedto form one unit dosage form. In some embodiments, a provided kitsfurther includes instructions for use.

Compounds and compositions described herein are generally useful for theinhibition of PRMT1. In some embodiments, methods of treatingPRMT1-mediated disorder in a subject are provided which compriseadministering an effective amount of a compound described herein (e.g.,a compound of Formula (I)), or a pharmaceutically acceptable saltthereof), to a subject in need of treatment. In certain embodiments, theeffective amount is a therapeutically effective amount. In certainembodiments, the effective amount is a prophylactically effectiveamount. In certain embodiments, the subject is suffering from aPRMT1-mediated disorder. In certain embodiments, the subject issusceptible to a PRMT1-mediated disorder.

As used herein, the term “PRMT1-mediated disorder” means any disease,disorder, or other pathological condition in which PRMT1 is known toplay a role. Accordingly, in some embodiments, the present disclosurerelates to treating or lessening the severity of one or more diseases inwhich PRMT1 is known to play a role.

In some embodiments, the present disclosure provides a method ofinhibiting PRMT1 comprising contacting PRMT1 with an effective amount ofa compound described herein (e.g., a compound of Formula (I)), or apharmaceutically acceptable salt thereof. The PRMT1 may be purified orcrude, and may be present in a cell, tissue, or subject. Thus, suchmethods encompass both inhibition of in vitro and in vivo PRMT1activity. In certain embodiments, the method is an in vitro method,e.g., such as an assay method. It will be understood by one of ordinaryskill in the art that inhibition of PRMT1 does not necessarily requirethat all of the PRMT1 be occupied by an inhibitor at once. Exemplarylevels of inhibition of PRMT1 include at least 10% inhibition, about 10%to about 25% inhibition, about 25% to about 50% inhibition, about 50% toabout 75% inhibition, at least 50% inhibition, at least 75% inhibition,about 80% inhibition, about 90% inhibition, and greater than 90%inhibition.

In some embodiments, provided is a method of inhibiting PRMT1 activityin a subject in need thereof (e.g., a subject diagnosed as having aPRMT1-mediated disorder) comprising administering to the subject aneffective amount of a compound described herein (e.g., a compound ofFormula (I)), or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof.

In certain embodiments, provided is a method of modulating geneexpression in a cell which comprises contacting a cell with an effectiveamount of a compound of Formula (I), or a pharmaceutically acceptablesalt thereof. In certain embodiments, the cell is in culture in vitro.In certain embodiments, the cell is in an animal, e.g., a human. Incertain embodiments, the cell is in a subject in need of treatment.

In certain embodiments, provided is a method of modulating transcriptionin a cell which comprises contacting a cell with an effective amount ofa compound of Formula (I), or a pharmaceutically acceptable saltthereof. In certain embodiments, the cell is in culture in vitro. Incertain embodiments, the cell is in an animal, e.g., a human. In certainembodiments, the cell is in a subject in need of treatment.

In certain embodiments, a method is provided of selecting a therapy fora subject having a disease associated with PRMT1-mediated disorder ormutation comprising the steps of determining the presence ofPRMT1-mediated disorder or gene mutation in the PRMT1 gene or andselecting, based on the presence of PRMT1-mediated disorder a genemutation in the PRMT1 gene a therapy that includes the administration ofa provided compound. In certain embodiments, the disease is cancer.

In certain embodiments, a method of treatment is provided for a subjectin need thereof comprising the steps of determining the presence ofPRMT1-mediated disorder or a gene mutation in the PRMT1 gene andtreating the subject in need thereof, based on the presence of aPRMT1-mediated disorder or gene mutation in the PRMT1 gene with atherapy that includes the administration of a provided compound. Incertain embodiments, the subject is a cancer patient. In someembodiments, a compound provided herein is useful in treating aproliferative disorder, such as cancer. For example, while not beingbound to any particular mechanism, protein arginine methylation by PRMT1is a modification that has been implicated in signal transduction, genetranscription, DNA repair and mRNA splicing, among others; andoverexpression of PRMT1 within these pathways is often associated withvarious cancers. Thus, compounds which inhibit the action of PRMT1, asprovided herein, are effective in the treatment of cancer.

In some embodiments, compounds provided herein are effective in treatingcancer through the inhibition of PRMT1. For example, PRMT1overexpression has been observed in various human cancers, including,but not limited to, breast cancer, prostate cancer, lung cancer, coloncancer, bladder cancer, and leukemia. In one example, PRMT1 specificallydeposits an asymmetric dimethylarginine (aDMA) mark on histone H4 atarginine 3 (H4R3me2a), and this mark is associated with transcriptionactivation. In prostate cancer, the methylation status of H4R3positively correlates with increasing tumor grade and can be used topredict the risk of prostate cancer recurrence (Seligson et al., Nature2005 435, 1262-1266). Thus, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with themethylation status of H4R3, e.g., prostate cancer. Additionally, themethylarginine effector molecule TDRD3 interacts with the H4R3me2a mark,and overexpression of TDRD3 is linked to poor prognosis for the survivalof patients with breast cancer (Nagahata et al., Cancer Sci. 2004 95,218-225). Thus, in some embodiments, inhibitors of PRMT1, as describedherein, are useful in treating cancers associated with overexpression ofTDRD3, e.g., breast cancer, as inhibition of PRMT1 leads to a decreasein methylation of H4R3, thereby preventing the association ofoverexpressed TDRD3 with H4R3me2a. In other examples, PRMT1 is known tohave non-histone substrates. For example, PRMT1, when localized to thecytoplasm, methylates proteins that are involved in signal transductionpathways, e.g., the estrogen receptor (ER). The expression status of ERin breast cancer is critical for prognosis of the disease, and bothgenomic and non-genomic ER pathways have been implicated in thepathogenesis of breast cancer. For example, it has been shown that PRMT1methylates ERα, and that ERα methylation is required for the assembly ofERα with SRC (a proto-oncogene tyrosine-protein kinase) and focaladhesion kinase (FAK). Further, the silencing of endogenous PRMT1resulted in the inability of estrogen to activate AKT. These resultssuggested that PRMT1-mediated ERα methylation is required for theactivation of the SRC-PI3K-FAK cascade and AKT, coordinating cellproliferation and survival. Thus, hypermethylation of ERα in breastcancer is thought to cause hyperactivation of this signaling pathway,providing a selective survival advantage to tumor cells (Le Romancer etal., Mol. Cell 2008 31, 212-221; Le Romancer et al., Steroids 2010 75,560-564). Accordingly, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with ERαmethylation, e.g., breast cancer. In yet another example, PRMT1 has beenshown to be involved in the regulation of leukemia development. Forexample, SRC-associated in mitosis 68 kDa protein (SAM68; also known asKHDRBS1) is a well-characterized PRMT1 substrate, and when either SAM68or PRMT1 is fused directly to the myeloid/lymphoid leukemia (MLL) gene,these fusion proteins can activate MLL oncogenic properties, implyingthat the methylation of SAM68 by PRMT1 is a critical signal for thedevelopment of leukemia (Cheung et al., Nature Cell Biol. 2007 9,1208-1215). Accordingly, in some embodiments, inhibitors of PRMT1, asdescribed herein, are useful in treating cancers associated with SAM68methylation, e.g., leukemia. In still another example, PRMT1 isimplicated in leukemia development through its interaction with AE9a, asplice isoform of AML1-ETO (Shia et al., Blood 2012 119:4953-62).Knockdown of PRMT1 affects expression of certain AE9a-activated genesand suppresses AE9a's self-renewal capability. It has also been shownthat AE9a recruits PRMT1 to AE9a activated gene promoters, which leadsto increased H4 Arg3 methylation, H3 Lys9/14 acetylation, andtranscription activated. Accordingly, in some embodiments, inhibitors ofPRMT1, as described herein, are useful in treating cancers associatedwith AML1-ETO, e.g., leukemia. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, is beneficial in the treatment of cancer.

In some embodiments, compounds described herein are useful for treatinga cancer including, but not limited to, acoustic neuroma,adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g.,lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g.,cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinomaof the breast, papillary carcinoma of the breast, mammary cancer,medullary carcinoma of the breast), brain cancer (e.g., meningioma;glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchuscancer, carcinoid tumor, cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colon cancer, rectal cancer, colorectaladenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma(e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma),endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma),familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g.,stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head andneck cancer (e.g., head and neck squamous cell carcinoma, oral cancer(e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g.,laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such asacute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acutemyelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma suchas Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkinlymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma(DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (e.g., “Waldenström's macroglobulinemia”), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease),hemangioblastoma, inflammatory myofibroblastic tumors, immunocyticamyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor,renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC),malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, smallcell lung cancer (SCLC), non-small cell lung cancer (NSCLC),adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma,myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV),essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocyticleukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilicsyndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis(NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g.,gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoidtumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma,pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer(e.g., Paget's disease of the penis and scrotum), pinealoma, primitiveneuroectodermal tumor (PNT), prostate cancer (e.g., prostateadenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer,skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g.,appendix cancer), soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous glandcarcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g.,seminoma, testicular embryonal carcinoma), thyroid cancer (e.g.,papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC),medullary thyroid cancer), urethral cancer, vaginal cancer and vulvarcancer (e.g., Paget's disease of the vulva).

In some embodiments, a compound provided herein is useful in treatingdiseases associated with increased levels of circulating asymmetricdimethylarginine (aDMA), e.g., cardiovascular disease, diabetes, kidneyfailure, renal disease, pulmonary disease, etc. Circulating aDMA isproduced by the proteolysis of asymmetrically dimethylated proteins.PRMT1 mediates aDMA methylation. aDMA levels are directly involved invarious diseases as aDMA is an endogenous competitive inhibitor ofnitric oxide synthase (NOS), thereby reducing the production of nitricoxide (NO) (Vallance et al., J. Cardiovasc. Pharmacol. 1992 20 (Suppl.12):S60-2). NO functions as a potent vasodilator in endothelial vessels,and as such inhibiting its production has major consequences on thecardiovascular system. For example, since PRMT1 is a major enzyme thatgenerates aDMA, the dysregulation of its activity is likely to regulatecardiovascular diseases (Boger et al., Ann. Med. 2006 38:126-36), andother pathophysiological conditions such as diabetes mellitus (Sydow etal., Vasc. Med. 2005 10(Suppl. 1):S35-43), kidney failure (Vallance etal., Lancet 1992 339:572-5), and chronic pulmonary diseases (Zakrzewiczet al., BMC Pulm. Med. 2009 9:5). Additionally, it has been demonstratedthat the expression of PRMT1 is increased in coronary heart disease(Chen et al., Basic Res. Cardiol. 2006 101:346-53). In another example,aDMA elevation is seen in patients with renal failure, due to impairedclearance of this metabolite from the circulation (Jacobi et al., Am. J.Nephrol. 2008 28:224-37). Thus, circulating aDMA levels is observed inmany pathophysiological situations. Accordingly, without being bound byany particular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, results in the decrease of circulating aDMA, which isbeneficial in the treatment of diseases associated with increased levelsof circulating aDMA, e.g., cardiovascular disease, diabetes, kidneyfailure, renal disease, pulmonary disease, etc. In certain embodiments,a compound described herein is useful for treating vascular diseases.

In some embodiments, a compound provided herein is useful in treatingmetabolic disorders. For example, PRMT1 has been shown to enhance mRNAlevels of FoxO1 target genes in gluconeogenesis, which results inincreased hepatic glucose production, and knockdown of PRMT1 promotesinhibition of FoxO1 activity and thus inhibition of hepaticgluconeogenesis (Choi et al., Hepatology 2012 56:1546-56). Additionally,genetic haploinsufficiency of Prmt1 has been shown to reduce bloodglucose levels in mouse models. Thus, without being bound by anyparticular mechanism, the inhibition of PRMT1, e.g., by compoundsdescribed herein, is beneficial in the treating of metabolic disorders,such as diabetes. In some embodiments, a provided compound is useful intreating type I diabetes. In some embodiments, a provided compound isuseful in treating type II diabetes.

In some embodiments, a compound provided herein is useful in treatingmuscular dystrophies. For example, PRMT1 methylates the nuclearpoly(A)-binding protein (PABPN1) in a region located near its C-terminus(Perreault et al., J. Biol. Chem. 2007 282:7552-62). This domain isinvolved in the aggregation of the PABPN1 protein, and abnormalaggregation of this protein is involved in the disease oculopharyngealmuscular dystrophy (Davies et al., Int. J. Biochem. Cell. Biol. 200638:1457-62). Thus, without being bound by any particular mechanism, theinhibition of PRMT1, e.g., by compounds described herein, is beneficialin the treatment of muscular dystrophies, e.g., oculopharyngeal musculardystrophy, by decreasing the amount of methylation of PABPN1, therebydecreasing the amount of PABPN1 aggregation.

In some embodiments, a compound provided herein is useful in treatingautoimmune disease. For example, several lines of evidence stronglysuggest that PRMT1 inhibitors may be valuable for the treatment ofautoimmune diseases, e.g., rheumatoid arthritis. PRMT1 is known tomodify and regulate several critical immunomodulatory proteins. Forexample, post-translational modifications (e.g., arginine methylation),within T cell receptor signaling cascades allow T lymphocytes toinitiate a rapid and appropriate immune response to pathogens.Co-engagement of the CD28 costimulatory receptor with the T cellreceptor elevates PRMT1 activity and cellular protein argininemethylation, including methylation of the guanine nucleotide exchangefactor Vav1 (Blanchet et al., J. Exp. Med. 2005 202:371-377). PRMT1inhibitors are thus expected to diminish methylation of the guanineexchange factor Vav1, resulting in diminished IL-2 production. PRMT1 isknown to enhance NFkB p65-driven transcription and facilitate thetranscription of p65 target genes like TNFα (Covic et al., Embo. J. 200524:85-96). Thus, in some embodiments, PRMT1 inhibitors, e.g., thosedescribed herein, are useful in treating autoimmune disease bydecreasing the transcription of p65 target genes like TNFα. Theseexamples demonstrate an important role for arginine methylation by PRMT1in inflammation. Thus, without being bound by any particular mechanism,the inhibition of PRMT1, e.g., by compounds described herein, isbeneficial in the treatment of autoimmune diseases.

In some embodiments, a compound provided herein is useful in treatingneurological disorders, such as amyotrophic lateral sclerosis (ALS). Forexample, a gene involved in ALS, TLS/FUS, often contains mutatedarginines in certain familial forms of this disease (Kwiatkowski et al.,Science 2009 323:1205-8). These mutants are retained in the cytoplasm,which is similar to reports documenting the role arginine methylationplays in nuclear-cytoplasmic shuffling (Shen et al., Genes Dev. 199812:679-91). This implicates PRMT1 function in this disease, as it wasdemonstrated that TLS/FUS is methylated on at least 20 arginine residues(Rappsilber et al., Anal. Chem. 2003 75:3107-14). Thus, in someembodiments, the inhibition of PRMT1, e.g., by compounds providedherein, are useful in treating ALS by decreasing the amount of TLS/FUSarginine methylation

Scheme 1 shows a general synthesis route to pyrazole compounds offormula I, wherein R^(y′) and R^(2′) are either the same as R^(y) and R²or are substituents which serve as precursors for synthetic conversionto R^(y) and R² wherein R², R^(y), R⁶, R^(x), R³, X, Y and Z and are asdefined above. In the first step iodopyrazole carboxaldehydes of generalformula XI are allowed to react with mono-Boc protected ethylenediaminesXII under reductive amination conditions (e.g. sodium cyanoborohydrideand catalytic acid such as acetic acid) in an appropriate solvent suchas methanol to give intermediates of general formula XIII. Suzukireaction of the latter with boronic acids or boronic esters of generalformula XIV in the presence of a palladium catalyst (e.g. PdCl₂(dppf))and a base (e.g. potassium carbonate) in an organic solvent (e.g.toluene) at elevated temperature yields intermediates of general formulaXV. In a subsequent optional step or steps, either R^(y′) or R^(2′) orboth R^(y′) and R^(2′) may be converted to yield R^(y) and R²substituents present in final compounds of formula I. For examplecompounds wherein R^(y) is —NHC(O)R^(A) or —NHSO₂R^(A) group can besynthesized by acylation or sulfonylation respectively of intermediatesof formula XV where R^(y′) is —NH₂ with a suitable acid chloride orsulfonylchloride in the presence of an appropriate base (e.g.diisopropylethylamine) in a suitable aprotic organic solvent (e.g.dichloromethane). In a final deprotection step the N-Boc protecting ofXV can be removed under a variety of conditions (e.g. HCl, TFA) in asuitable organic solvent (e.g. ethanol, THF, dichloromethane) to givecompounds of formula I. In certain embodiments, compounds wherein R^(y)is (CR^(z)R^(z))_(n)C(O)N(R^(B))₂ group can be synthesized by amidecoupling reaction of intermediates of formula XV where R^(y′) is —CO₂Hwith a an amine of formula HN(R^(B))₂.

In certain embodiments, iodopyrazole carboxaldehydes of general formulaXI are prepared from suitable known pyrazole compound intermediates byestablished synthetic chemistry methods. Standard methods include directiodination of a pyrazole 3-carboxylate and Sandmeyer reaction of a3-amino pyrazole 4-carboxylate. In certain embodiments, iodopyrazolecarboxaldehydes are derived from iodopyrazole carboxylates by reductionto a hydroxymethyl group followed by oxidation to carboxaldehyde. Incertain embodiments, mono-Boc protected ethylenediamines XII can besynthesized by standard methods known in the literature for derivatizingor preparing ethylenediamines. For example intermediates of formula XIImay be prepared by treatment of the corresponding unprotected diamineprecursors with Boc₂O and purifying the mixture of mono and dibocylatedproducts. Phenyl boronic acids or esters of general formula XIV areeither commercially available or can be prepared by standard method forexample by boronylation of the corresponding commercially availablephenylbromides. In certain embodiments, pyrazole compounds of generalformula II are prepared from iodopyrazole carboxaldehydes of generalformula XXI as depicted in Scheme 2. In certain embodiments where R⁴ ishydrogen compounds of general formula II are equivalent to compounds ofgeneral formula III which are tautomers. In these cases R^(4′) is aprotecting group such as tetrahydropyranyl (THP) which maybe cleaved tohydrogen under acidic conditions in the final Boc-deprotection step. Inone synthetic route iodopyrazole carboxaldehydes of general formula XXIcan be prepared as depicted in Scheme 3.

In certain embodiments, iodopyrazole carboxaldehydes of general formulaXXI are prepared as depicted in Scheme 4 which also providesiodopyrazole carboxaldehydes of general formula XXXI. Thus, alkylationof intermediates of general formula XXX gives a mixture of pyrazolenitrogen alkylated isomers which are separated by chromatography to givepure isomers XXI and XXXI. In certain embodiments, pyrazole compounds ofgeneral formula III can be prepared from iodopyrazole carboxaldehydes ofgeneral formula XXXI as depicted in Scheme 5.

In certain embodiments, pyrazole compounds of general formula IV areprepared from iodopyrazole carboxaldehydes of general formula XLI asdepicted in Scheme 6. In certain embodiments where R⁴ is hydrogencompounds of general formula IV are equivalent to compounds of generalformula V which are tautomers. In certain embodiments where R⁴ incompounds of formula IV is hydrogen, R^(4′) in intermediate XLI may be aselected protecting group such as tetrahydropyranyl (THP) which can becleaved to hydrogen under acidic conditions in the finalBoc-deprotection step.

In certain embodiments, iodopyrazole carboxaldehydes of general formulaXLI and LI are prepared as depicted in Scheme 7. In certain embodimentsan R⁴ group of iodopyrazole carboxaldehydes is introduced by alkylationof intermediates of formula XLVII. This reaction can give a mixture ofintermediate compounds of formulas XLI and LI which may be separated bychromatography. The THP protected intermediates of formula XLVI can beused to prepare compounds of formula IV where R⁴═H as also depicted inScheme 7.

In certain embodiments, pyrazole compounds of general formula V can beprepared from iodopyrazole carboxaldehydes of general formula LI asdepicted in Scheme 8.

In certain embodiments, phenyl boronic acids or esters of generalformula XIV are commercially available or can be prepared fromcommercially available or known corresponding phenyl bromide precursorsof general formula LX by standard methods. One such method to preparepinacol boranes XIVd is depicted in Scheme 9.

In certain embodiments, compounds of formula XIV are prepared bymultistep organic synthesis using known methods. For example bromides LXmay be prepared from corresponding aniline intermediate compounds bySandmeyer reaction. The required aniline intermediates can in turn beprepared from the corresponding nitrobenzenes by reduction of the nitrogroup under standard conditions (e.g. Pd/H₂, SnCl₂, Fe/HOAc).Alternatively certain embodiments of compounds of formula XIVd may beeffectively prepared by iridium catalyzed borylation (Hartwig, J. et.Al. Journal of the American Chemical Society 2005, 127, 14278) ofintermediates LXX as depicted in Scheme 10.

Examples

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Synthetic Methods

General methods and experimental procedures for preparing andcharacterizing compounds of the present invention are set forth below.Wherever needed, reactions were heated using conventional hotplateapparatus or heating mantle or microwave irradiation equipment.Reactions were conducted with or without stirring, under atmospheric orelevated pressure in either open or closed vessels. Reaction progresswas monitored using conventional techniques such as TLC, HPLC, UPLC, orLCMS using instrumentation and methods described below. Reactions werequenched and crude compounds isolated using conventional methods asdescribed in the specific examples provided. Solvent removal was carriedout with or without heating, under atmospheric or reduced pressure,using either a rotary or centrifugal evaporator. Compound purificationwas carried out as needed using a variety of traditional methodsincluding, but not limited to, preparative chromatography under acidic,neutral, or basic conditions using either normal phase or reverse phaseHPLC or flash columns or Prep-TLC plates. Compound purity and massconfirmations were conducted using standard HPLC and/or UPLC and/or MSspectrometers and/or LCMS and/or GC equipment (e.g., including, but notlimited to the following instrumentation: Waters Alliance 2695 with 2996PDA detector connected with ZQ detector and ESI source; ShimadzuLDMS-2020; Waters Acquity H Class with PDA detector connected with SQdetector and ESI source; Agilent 1100 Series with PDA detector; WatersAlliance 2695 with 2998 PDA detector; AB SCIEX API 2000 with ESI source;Agilent 7890 GC). Exemplified compounds were dissolved in either MeOH orMeCN to a concentration of approximately 1 mg/mL and analyzed byinjection of 0.5-10 μL into an appropriate LCMS system using the methodsprovided in the following table:

MS Heat MS Block Detector Mobile Mobile Flow Rate Temp Voltage MethodColumn Phase A Phase B (mL/min) Gradient Profile (° C.) (kV) A Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 2.0 250 1.5 XR-ODS TFA TFAminutes, 100% B for 1.1 2.2 μm minutes, 100% to 5% B in 3.0 × 50 mm 0.2minutes, then stop B Gemini-NX Water/0.04% ACN 1 5% to 100% B in 2.0 2000.75 3 μm C18 Ammonia minutes, 100% B for 1.1 110A minutes, 100% to 5% Bin 0.1 minutes, then stop C Shim-pack Water/0.05% ACN/0.05% 1 5% to 100%B in 2.0 250 0.85 XR-ODS FA FA minutes, 100% B for 1.1 1.6 μm minutes,100% to 5% B in 2.0 × 50 mm 0.1 minutes, then stop D Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 2.0 250 0.95 XR-ODS TFA TFAminutes, 100% B for 1.1 2.2 μm minutes, 100% to 5% B in 3.0 × 50 mm 0.1minutes, then stop E Waters Water/0.05% ACN/0.05% 0.9 5% to 100% B in2.0 250 1.5 Xselect C18 FA FA minutes, 100% B for 1.2 3.5 μm minutes,100% to 5% B in 3.0 × 50 mm 0.1 minutes, then stop F Shim-packWater/0.05% ACN/0.05% 1 5% to 80% B in 3.25 200 0.95 XR-ODS TFA TFAminutes, 80% B for 1.35 2.2 μm minutes, 80% to 5% B in 3.0 × 50 mm 0.3minutes, then stop G Shim-pack Water/0.05% ACN/0.05% 1 5% to 70% B in2.50 200 0.95 XR-ODS TFA TFA minutes, 70% B for 0.70 2.2 μm minutes, 70%to 5% B in 3.0 × 50 mm 0.1 minutes, then stop H Shim-pack Water/0.05%ACN/0.05% 1 5% to 100% B in 2.20 250 0.95 XR-ODS TFA TFA minutes, 100% Bfor 1.00 2.2 μm minutes, 100% to 5% B in 3.0 × 50 mm 0.1 minutes, thenstop I Shim-pack Water/0.05% ACN/0.05% 1 5% to 100% B in 1.20 250 0.95XR-ODS TFA TFA minutes, 100% B for 1.00 2.2 μm minutes, 100% to 5% B in3.0 × 50 mm 0.1 minutes, then stop J Shim-pack Water/0.05% ACN/0.05% 15% to 70% B in 3.20 250 0.95 XR-ODS TFA TFA minutes, 70% B for 0.75 2.2μm minutes, 70% to 5% B in 3.0 × 50 mm 0.35 minutes, then stop KShim-pack Water/0.05% ACN/0.05% 1 5% to 80% B in 3.00 250 1.5 XR-ODS TFATFA minutes, 80% B for 0.8 2.2 μm minutes, 80% to 5% B in 3.0 × 50 mm0.1 minutes, then stop L Shim-pack Water/0.05% ACN/0.05% 1 5% to 100% Bin 3.00 250 1.5 XR-ODS TFA TFA minutes, 100% B for 0.8 2.2 μm minutes,100% to 5% B in 3.0 × 50 mm 0.1 minutes, then stop M Shim-packWater/0.05% ACN/0.05% 1 5% to 100% B in 2.20 250 1.5 XR-ODS TFA TFAminutes, 100% B for 1.00 2.2 μm minutes, 100% to 5% B in 3.0 × 50 mm 0.1minutes, then stop N Shim-pack Water/0.05% ACN/0.05% 1 5% to 80% B in2.20 250 1.5 XR-ODS TFA TFA minutes, 80% B for 1.0 2.2 μm minutes, 80%to 5% B in 3.0 × 50 mm 0.1 minutes, then stop O Zorbax Water/0.05%ACN/0.05% 1 5% to 70% B in 8.00 250 1.5 Eclipse Plus TFA TFA minutes,70% B for 2.0 C18 minutes, then stop 4.6 × 100 mm P Shim-packWater/0.05% ACN/0.05% 1 5% to 65% B in 3.00 250 1.5 XR-ODS TFA TFAminutes, 65% B for 0.80 2.2 μm minutes, 100% to 5% B in 3.0 × 50 mm 0.1minutes, then stop Q Shim-pack Water/0.05% ACN/0.05% 1 5% to 60% B in2.50 250 0.95 XR-ODS TFA TFA minutes, 60% B for 0.7 2.2 μm minutes, 60%to 5% B in 3.0 × 50 mm 0.1 minutes, then stop R Shim-pack Water/0.05%ACN/0.05% 1 5% to 50% B in 2.50 250 0.95 XR-ODS TFA TFA minutes, 50% Bfor 0.7 3.0 × 50 mm minutes, 50% to 5% B in 0.1 minutes, then stop SXBridge Water/0.05% ACN/0.05% 1 5% to 95% B in 2.20 250 0.9 C18 3.5 μmTFA TFA minutes, 95% B for 1.00 3.0 × 50 mm minutes, 95% to 5% B in 0.1minutes, then stop T Shim-pack Water/0.05% ACN/0.05% 0.7 5% to 100% B in2.0 250 0.85 XR-ODS FA FA minutes, 100% B for 1.1 1.6 μm minutes, 100%to 5% B in 2.0 × 50 mm 0.1 minutes, then stop U Shim-pack Water/0.05%ACN/0.05% 1 5% to 40% B in 2.50 250 0.95 XR-ODS TFA TFA minutes, 40% Bfor 0.7 2.2 μm minutes, 40% to 5% B in 3.0 × 50 mm 0.1 minutes, thenstop V Shim-pack Water/0.05% ACN/0.05% 1 5% to 60% B in 4.20 200 1.05XR-ODS TFA TFA minutes, 60% B for 1.0 2.2 μm minutes, 60% to 5% B in 3.0× 50 mm 0.1 minutes, then stop W Shim-pack Water/0.05% ACN/0.05% 1 5% to100% B in 2.20 200 0.95 XR-ODS TFA TFA minutes, 100% B for 1.00 2.2 μmminutes, 100% to 5% B in 3.0 × 50 mm 0.1 minutes, then stop X Shim-packWater/0.05% ACN/0.05% 0.7 5% to 100% B in 2.0 200 0.85 XR-ODS FA FAminutes, 100% B for 1.1 1.6 μm minutes, 100% to 5% B in 2.0 × 50 mm 0.1minutes, then stop Y Ecliplis Plus Water/0.05% ACN 1 5% to 100% B in 2.0250 1 C18 3.5 μm TFA minutes, 100% B for 1.0 4.6 × 50 mm minutes, 100%to 5% B in 0.1 minutes, then stop Z Ecliplis Plus Water/10 ACN/5% 1 5%to 100% B in 2.0 250 1.1 C18 3.5 μm mM water minutes, 100% B for 1.0 4.6× 50 mm ammonium minutes, 100% to 5% B in carbonate 0.1 minutes, thenstop Al Shim-pack Water/0.05% ACN 1 5% to 100% B in 2.0 250 1 XR-ODS TFAminutes, 100% B for 1.0 2.2 μm minutes, 100% to 5% B in 3.0 × 50 mm 0.1minutes, then stop A2 Ecliplis Plus Water/10 ACN 1 5% to 100% B in 2.0250 0.95 C18 3.5 μm mM minutes, 100% B for 1.4 4.6 × 50 mm ammoniumminutes, 100% to 5% B in acetate 0.1 minutes, then stop A3 AcquityWater/5 mM ACN/0.1% 0.55 5% B at 0.01 min up to 0.4 BEH C18 ammonium FAmin, 35% B at 0.8 min, 55% 1.7 μm acetate/ B at 1.2 min, 100% B in 1.32.1 × 50 mm 0.1% FA minutes, at 2.5 min up to 3.30 min, 5% B at 3.31 minup to 4.0 min, then stop

Compound structure confirmations were carried out using standard 300 or400 MHz NMR spectrometers with NOe's conducted whenever necessary.

The following abbreviations are used herein:

Abbreviation Meaning ACN acetonitrile atm. atmosphere DCMdichloromethane DHP dihydropyran DIBAL diisobutyl aluminum hydride DIEAdiisopropyl ethylamine DMF dimethyl formamide DMF-DMA dimethyl formamidedimethyl acetal DMSO dimethyl sulfoxide dppf1,1′-bis(diphenylphosphino)ferrocene EA ethyl acetate ESI electrosprayionization EtOH ethanol FA formic acid GC gas chromatography h hour Hexhexanes HMDS hexamethyl disilazide HPLC high performance liquidchromatography IPA isopropanol LCMS liquid chromatography/massspectrometry MeOH methanol min minutes NBS N-bromo succinimide NCSN-chloro succinimide NIS N-iodo succinimide NMR nuclear magneticresonance NOe nuclear Overhauser effect Prep. preparative PTSApara-toluene sulfonic acid Rf retardation factor rt room temperature RTretention time sat. saturated SGC silica gel chromatography TBAFtetrabutyl ammonium fluoride TEA triethylamine TFA trifluoroacetic acidTHF tetrahydrofuran TLC thin layer chromatography UPLC ultra performanceliquid chromatography

Intermediate Synthesis Synthesis of intermediate tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

Step 1: tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (3.2 g,10.45 mmol, 1.00 equiv), tert-butyl N-[2-(methylamino)ethyl]carbamate(2.2 g, 12.63 mmol, 1.21 equiv) and NaBH(OAc)₃ (6.65 g, 31.38 mmol, 3.00equiv) in dichloroethane (30 mL) was stirred for 2 h at roomtemperature. The reaction was quenched with 50 mL of saturated aqueoussodium bicarbonate solution. The resulting mixture was extracted with3×200 mL of dichloromethane. The combined organic layers was dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluted with 30-100% ethyl acetate inpetroleum ether to give 4.05 g (83%) of tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamateas a light yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.48 (s, 1H),5.35-5.30 (m, 1H), 4.13-4.03 (m, 1H), 3.71-3.63 (m, 1H), 3.36 (s, 2H),3.26-3.25 (m, 2H), 2.52-2.49 (m, 2H), 2.21 (s, 3H), 2.09-2.01 (m, 3H),1.68-1.58 (m, 3H), 1.44 (s, 9H) ppm. LCMS (method C, ESI): RT=0.58 min,m/z=465.0 [M+H]⁺.

Synthesis of intermediate tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

Step 1: Ethyl 3-iodo-1H-pyrazole-4-carboxylate

To a stirred solution of ethyl 3-amino-1H-pyrazole-4-carboxylate (10 g,64.45 mmol, 1.00 equiv) in 50% sulfuric acid (90 mL) at 5° C. was addeddropwise a solution of NaNO₂ (7.4 g, 107.25 mmol, 1.66 equiv) in water(15 mL). The reaction was stirred at 5° C. for another 30 min. Asolution of KI (32.1 g, 193.37 mmol, 3.00 equiv) in water (15 mL) wasadded dropwise at 5° C. The reaction was allowed to stir at 5° C. for 1h and then quenched by the addition of 50 mL of water. The precipitatewas collected by filtration and then dissolved in 150 mL of ethylacetate. The resulting solution was washed sequentially with 1×100 mL ofsaturated Na₂SO₃ solution, 1×100 mL of saturated sodium bicarbonatesolution and 1×100 mL of brine. The organic layer was dried overanhydrous sodium sulfate and concentrated under vacuum to give 10.8 g(63%) of ethyl 3-iodo-1H-pyrazole-4-carboxylate as a yellow solid. ¹HNMR (300 MHz, CDCl₃): δ 8.18 (s, 1H), 4.38-4.29 (m, 2H), 1.41-1.33 (m,3H) ppm. LCMS (method B, ESI): RT=1.36 min, m/z=267.0 [M+H]⁺.

Step 2: Ethyl3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylate

A solution of ethyl 3-iodo-1H-pyrazole-4-carboxylate (10.8 g, 40.60mmol, 1.00 equiv), 3,4-dihydro-2H-pyran (10 g, 118.88 mmol, 2.93 equiv)and TsOH (780 mg, 4.53 mmol, 0.11 equiv) in THF (100 mL) was stirred for2 h at 60° C. The reaction mixture was cooled to room temperature andquenched by the addition of 100 mL of saturated sodium bicarbonatesolution. The resulting solution was extracted with 2×80 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with ethyl acetate/petroleum ether (1:20)to give 13 g (91%) of ethyl3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate as a yellow oil. ¹H NMR(400 MHz, CDCl₃): δ 8.04 (s, 1H), 5.40-5.38 (m, 1H), 4.34-4.29 (m, 2H),4.08-4.05 (m, 1H), 3.73-3.70 (m, 1H), 2.07-1.98 (m, 3H), 1.69-1.62 (m,3H), 1.39-1.32 (m, 3H) ppm. LCMS (method C, ESI): RT=1.53 min, m/z=351.0[M+H]⁺.

Step 3: 3-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carboxylicacid

To a solution of ethyl 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylate(85 g, 242.75 mmol, 1.00 equiv) in THF (300 mL) and methanol (300 mL)was added a solution of LiOH (17.5 g, 730.69 mmol, 3.01 equiv) in water(400 mL). The resulting solution was stirred at room temperatureovernight and then concentrated under vacuum to remove the organicsolvent. The resulting solution was diluted with 400 mL of H₂O and thenacidified to pH 6.0 with 1M hydrochloric acid. The mixture was extractedwith 3×800 mL of dichloromethane. The combined organic layers was washedwith 3×1000 mL of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum to give 75 g (96%) of3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid as an off-whitesolid. LCMS (method D, ESI): RT=1.23 min, m/z=323.0 [M+H]⁺.

Step 4: (3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanol

To a solution of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carboxylic acid (28g, 86.93 mmol, 1.00 equiv) in anhydrous THF (300 mL) maintained undernitrogen at 5° C. was added a 1M solution of BH₃ in THF (300 mL)dropwise with stirring. The reaction was stirred overnight at roomtemperature and then quenched by the addition of 300 mL of saturatedNH₄Cl solution. The resulting mixture was extracted with 3×1000 mL ofdichloromethane. The combined organic layers was dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluted with ethyl acetate/petroleum ether (1:1)to give 12.67 g (47%) of (3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanolas a white solid. ¹H NMR (400 MHz, DMSO-d6): δ 7.73 (s, 1H), 5.37-5.34(m, 1H), 4.92 (s, 1H), 4.20 (d, J=3.6 Hz, 2H), 3.89-3.88 (m, 1H),3.65-3.57 (m, 1H), 2.09-2.00 (m, 1H), 1.99-1.90 (m, 2H), 1.69-1.61 (m,1H), 1.49-1.46 (m, 2H) ppm. LCMS (method A, ESI): RT=1.16 min, m/z=309.0[M+H]⁺.

Step 5: 3-Iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde

Into a 250-mL 3-necked round-bottom flask purged and. To a stirredsolution of oxalyl chloride (18.576 g, 146.35 mmol, 3.01 equiv) inanhydrous dichloromethane (300 mL) maintained under nitrogen at −78° C.was added DMSO (15.138 g, 193.75 mmol, 3.98 equiv) dropwise. Thereaction mixture was stirred at −65° C. for 30 min. A solution of(3-iodo-1-(oxan-2-yl)-1H-pyrazol-4-yl)methanol (15.0 g, 48.68 mmol, 1.00equiv) in dichloromethane (100 mL) was then added dropwise at −65° C.and the reaction was stirred for another 60 min at −65° C. Triethylamine(40.6 mL) was added dropwise at −65° C. and the reaction was stirred for30 min at −65° C. The reaction was warmed to 0° C. then quenched by theaddition of 100 mL of saturated NH₄Cl solution. The resulting mixturewas extracted with 3×400 mL of dichloromethane. The combined organiclayers was dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluted withethyl acetate/petroleum ether (1:20) to give 13.48 g (90%) of3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde as a golden oil. ¹H NMR(300 MHz, DMSO-d6): δ 9.69 (s, 1H), 8.57 (s, 1H), 5.49 (dd, J=2.7 Hz,9.9 Hz, 1H), 3.95-3.91 (m, 1H), 3.68-3.62 (m, 1H), 2.11-2.01 (m, 3H),1.69-1.62 (m, 3H) ppm.

LCMS (method A, ESI): RT=1.35 min, m/z=307.0 [M+H]⁺.

Step 6: tert-Butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

A mixture of 3-iodo-1-(oxan-2-yl)-1H-pyrazole-4-carbaldehyde (21.5 g,70.24 mmol, 1.00 equiv), tert-butylN-methyl-N-(2-(methylamino)ethyl)carbamate (20 g, 106.23 mmol, 1.51equiv) and NaBH(OAc)₃ (29.8 g, 137.98 mmol, 1.96 equiv) indichloroethane (300 mL) was stirred for 1 h at room temperature. Thereaction was diluted with 300 mL of dichloromethane and then washed with3×300 mL of brine. The organic layer was dried over anhydrous sodiumsulfate and concentrated under vacuum. The residue was purified on asilica gel column eluted with 0-7% methanol in dichloromethane to give31 g (92%) of tert-butyl(2-(((3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamateas a yellow oil. ¹H NMR (300 MHz, CDCl₃): δ 7.62 (s, 1H), 5.34-5.30 (m,1H), 4.06-4.02 (m, 1H), 3.68-3.62 (m, 1H), 3.42-3.38 (m, 4H), 2.85 (s,4H), 2.62-2.53 (m, 2H), 2.47-2.46 (m, 2H), 2.13-1.97 (m, 3H), 1.74-1.69(m, 3H), 1.46 (s, 9H) ppm. LCMS (method A, ESI): RT=1.17 min, m/z=479.0[M+H]⁺.

Compound 1N-(4-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1H-pyrazol-3-yl)phenyl)methacrylamide

Step 1:3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde

To a solution of3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (1 g,0.00326 mol) in 1,4-dioxane (30 ml) were added 4-nitrophenylboronic acid(0.654 g, 0.00392 mol) and sat. NaHCO₃ solution (8 ml). The reactionmixture was degassed using argon for 0.5 h. Tetrakis(triphenylphosphine)palladium (0.188 g, 0.000163 mol) was added under an argon atm. and theresultant suspension was heated for 8 h at 90° C. The reaction wascooled to room temperature, poured into water (100 mL) and extractedwith EtOAc (100 mL×3). The combined extracts was washed with brine (100mL), dried over sodium sulphate and evaporated under reduced pressure.The residue was purified by SGC using hexane: EtOAc (0%-40%) as eluentto obtain3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde(550 mg, Yield 56%). LCMS (ESI): RT=2.32 min, m/z=302.2 [M+H]⁺.

Step 2: tert-butylmethyl(2-(methyl((3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)amino)ethyl)carbamate

To a solution of3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde(0.550 g, 0.00182 mol) in methanol (10 mL) at room temperature wereadded TEA (221 mg, 0.00913 mol), ZnCl₂ (0.025 g, 0.000182 mol) andtert-butyl methyl(2-(methylamino)ethyl)carbamate (0.410 g, 0.00219 mol)and stirred for 45 min. NaCNBH₃ (0.580 g, 0.00913 mol) was added and thereaction mixture was stirred for 2 h at room temperature. The reactionwas poured into water (70 mL) and extracted with EtOAc (70 mL×2). Thecombined extracts were washed with brine (100 mL), dried over sodiumsulphate and concentrated under vacuum. The residue was purified by SGCusing DCM: MeOH (0%-4%) as eluent to obtain tert-butylmethyl(2-(methyl((3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)amino)ethyl)carbamate(0.770 g, Yield: 86.71%). LCMS (ESI): RT=2.00 min, m/z=474.8 [M+H]⁺.

Step 3: tert-butyl(2-(((3-(4-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

To a solution of tert-butylmethyl(2-(methyl((3-(4-nitrophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)amino)ethyl)carbamate(0.700 g, 0.00140 mol) in MeOH (25 mL) was added Pd/C (0.07 g, 10% w/w)under a nitrogen atmosphere. Hydrogen gas was purged into the reactionmass for 2 h. The reaction mixture was filtered through a Celite bed andwashed with MeOH. The filtrate was evaporated to give tert-butyl(2-(((3-(4-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(0.600 g, yield: 92%). LCMS (ESI): RT=1.79 min, m/z=444.5 [M+H]⁺.

Step 4: tert-butyl(2-(((3-(4-methacrylamidophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

A solution of tert-butyl(2-(((3-(4-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(0.150 g, 0.000338 mol) in DCM (3 mL) was cooled to 0° C. TEA (0.068 g,0.000677 mol) and methacryloyl chloride (0.042 g, 0.000406 mol) wereadded and the reaction mixture stirred for 15 mins at 0° C. The reactionwas poured into water (25 mL) and extracted with EtOAc (20 mL×2). Thecombined extracts was washed with brine (30 mL), dried over sodiumsulphate and concentrated under vacuum. The residue was purified by SGCusing DCM:MeOH (0%-5%) as eluent to obtain tert-butyl(2-(((3-(4-methacrylamidophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(0.080 g, Yield: 45%). ESI-MS: m/z=512.3 [M+1]⁺.

Step 5:N-(4-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1H-pyrazol-3-yl)phenyl)methacrylamide(Compound 1)

A solution of tert-butyl(2-(((3-(4-methacrylamidophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(0.08 g, 0.00015 mol) in methanolic HCl (2 mL, 4M solution) was stirredfor 1 hr at room temperature. The reaction mixture was concentratedunder vacuum and purified by prep HPLC using water: ACN as mobile phase(containing 0.1% TFA as modifier) to obtainN-(4-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1H-pyrazol-3-yl)phenyl)methacrylamide(0.040 g, yield: 80%). ¹H NMR (400 MHz, CD₃OD): δ 7.98 (s, 1H),7.84-7.81 (m, 2H), 7.59-7.56 (d, 2H), 5.85 (s, 1H) 5.58 (s, 1H), 5.52(s, 1H), 4.51 (s, 2H), 3.37 (s), 2.72 (s, 3H), 2.70 (s, 3H), 2.06 (s,3H) ppm; LCMS (ESI): RT=1.63 min, m/z=328.3 [M+H]⁺.

Compound 2N-(4-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1H-pyrazol-3-yl)phenyl)acrylamide

Step 1: tert-butyl2-(((3-(4-acrylamidophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate

A solution of tert-butyl2-(((3-(4-aminophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)carbamate(150 mg, 0.33 mmol) in DCM (2 mL) and TEA (68 mg, 0.66 mmol) was stirredat 0° C. followed by dropwise addition of acryloyl chloride (37 mg, 0.39mmol). The reaction mixture was stirred for 10 minutes then poured intowater (20 mL), and extracted with EtOAc (15 mL×2). The combined extractswas washed with brine (20 mL), dried over sodium sulphate andconcentrated under vacuum. The residue was purified by SGC using DCM:MeOH (0%-7%) as eluent to obtain tert-butyl2-(((3-(4-acrylamidophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(55 mg, Yield 34%).

Step 2:N-(4-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1H-pyrazol-3-yl)phenyl)acrylamide(Compound 2)

A solution of tert-butyl2-(((3-(4-acrylamidophenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)(methyl)amino)ethyl)(methyl)carbamate(55 mg, 0.0001 mmol) in DCM (2 mL) and TFA (0.5 mL) was stirred for 2 h.The solvent was evaporated and the residue was purified by triturationwith diethyl ether to obtain title compound (40 mg, Yield 92% as aTFAsalt). ¹H NMR (400 MHz, CD₃OD): δ 7.93 (s, 1H), 7.84 (d, J=8.0 Hz, 2H),7.60 (d, J=8.0 Hz, 2H), 6.50-6.40 (m, 2H), 5.84 (d, J=9.2, 1H), 4.38 (s,2H), 3.55-3.45 (m, 1H), 3.25-3.15 (s, 2H), 2.69 (s, 3H), 2.66 (s, 3H)ppm. LCMS (ESI): RT=7.57 min, m/z=314.0 [M+H]⁺.

Compound 3 3-methyl-N-(3-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1H-pyrazol-3-yl)phenyl)but-2-enamide

Step 1:3-methyl-N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)but-2-enamide

To a solution of 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline(1.0 g, 0.00456 mol) in THF (5 mL) at 0° C., were added TEA (0.552 g,0.000547 mol) and dimethylacryloyl chloride (0.648 g, 0.00456 mol) andstirred for 15 mins at 0° C. The reaction was poured into water (200 mL)and extracted with EtOAc (300 mL×2). The combined extracts was washedwith brine (200 mL), dried over sodium sulphate, and concentrated undervacuum to afford3-methyl-N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)but-2-enamide(0.7 g, Yield: 51%).

Step 2:N-(3-(4-formyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)phenyl)-3-methylbut-2-enamide

To a solution of3-methyl-N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)but-2-enamide(0.261 g, 0.00098 mol) and3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-4-carbaldehyde (0.250 g,0.000817 mol) in 1,4-dioxane (8 ml) was added saturated NaHCO₃ solution(8 mL). The reaction mixture was degassed using argon for 0.5 h.Tetrakis(triphenylphosphine) palladium (0.094 g, 0.000081 mol) was addedunder an argon atm. and the resultant suspension was heated for 8 h at90° C. The reaction was poured into water (50 mL) extracted with EtOAc(40 mL×2). The combined extracts were washed with brine (50 mL) driedover sodium sulphate and concentrated under vacuum. The residue waspurified by SGC using DCM:MeOH (0-1%) as eluent to obtainN-(3-(4-formyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)phenyl)-3-methylbut-2-enamide(0.210 g, Yield 73.43%). LCMS (ESI): RT=2.20 min, m/z=354.3 [M+H]⁺.

Step 3: tert-butylmethyl(2-(methyl((3-(3-(3-methylbut-2-enamido)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)amino)ethyl)carbamate

To a solution ofN-(3-(4-formyl-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)phenyl)-3-methylbut-2-enamide(0.210 g, 0.00062 mol) in methanol (4 mL) were added TEA (313 mg, 0.0031mol), ZnCl₂ (0.008 g, 0.000062 mol) and tert-butylmethyl(2(methylamino)ethyl)carbamate (0.138 g, 0.00074 mol). Thereaction mixture was stirred at rt for 45 min. NaCNBH₃ (0.580 g, 0.00913mol) was added and stirred for 2 h at rt. The reaction was poured intowater (100 mL) and extracted with EtOAc (100 mL×2). The combinedextracts was washed with brine (200 mL), dried over sodium sulphate andconcentrated under vacuum. The residue was purified by SGC usingDCM:MeOH (0%-4%) as eluent to obtain tert-butylmethyl(2-(methyl((3-(3-(3-methylbut-2-enamido)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)amino)ethyl)carbamate(0.150 g, Yield: 47%). LCMS (ESI): RT=2.03 min, m/z=526.5 [M+H]⁺.

Step 4: 3-methyl-N-(3-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)phenyl)but-2-enamide(Compound 3)

A solution of tert-butylmethyl(2-(methyl((3(3-3-methylbut-2-enamido)phenyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methyl)amino)ethyl)carbamate(0.08 g, 0.000228 mol) in methanolic HCl (2 mL, 4M) was stirred for 1 hrat room temperature. The solution was concentrated under reduce pressureand triturated with diethyl ether to give 3-methyl-N-(3-(4-((methyl(2-(methylamino)ethyl)amino)methyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-3-yl)phenyl)but-2-enamide (0.026 g, yield: 33%, HCl salt). ¹H NMR (400 MHz,DMSO-d6): δ 8.02-7.80 (m, 2H), 7.60-7.50 (m, 1H), 7.44-7.35 (m, 1H),7.30-7.21 (m, 1H), 5.90 (s, 1H), 4.50-4.20 (s, 2H), 3.30-3.00 (m), 2.60(s, 3H), 2.52 (s, 3H), 2.15 (s, 3H), 1.88 (s, 3H) ppm. LCMS (ESI):RT=1.49 min, m/z=342.4 [M+H]⁺.

Biological Methods

PRMT1 Biochemical Assay

General Materials.

S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), bicine,Tween20, dimethylsulfoxide (DMSO), bovine skin gelatin (BSG), andTris(2-carboxyethyl)phosphine hydrochloride solution (TCEP) werepurchased from Sigma-Aldrich at the highest level of purity possible.³H-SAM was purchase from American Radiolabeled Chemicals with a specificactivity of 80 Ci/mmol. 384-well streptavidin Flashplates were purchasedfrom PerkinElmer.

Substrates.

Peptide representative of human histone H4 residues 36-50 wassynthesized with an N-terminal linker-affinity tag motif and aC-terminal amide cap by 21^(st) Century Biochemicals. The peptide waspurified by high-performance liquid chromatography (HPLC) to greaterthan 95% purity and confirmed by liquid chromatography mass spectrometry(LC-MS). The sequence was Biot-Ahx-RLARRGGVKRISGLI-amide (SEQ ID NO.:1).

Molecular Biology:

Full-length human PRMT1 isoform 1 (NM_001536.5) transcript clone wasamplified from an HEK 293 cDNA library, incorporating flanking 5′sequence encoding a FLAG tag (DYKDDDDK) (SEQ ID NO.:2) fused directly toMet 1 of PRMT1. The amplified gene was subcloned into pFastBacI (LifeTechnologies) modified to encode an N-terminal GST tag and a TEVcleavage sequence(MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYFQGGNS) (SEQ ID NO.:3)fused to Asp of the Flag tag of PRMT1.

Protein Expression.

Recombinant baculovirus were generated according to Bac-to-Bac kitinstructions (Life Technologies). Protein over-expression wasaccomplished by infecting exponentially growing High Five insect cellculture at 1.5×10⁶ cell/ml with 1:100 ratio of virus. Infections werecarried out at 27° C. for 48 hours, harvested by centrifugation, andstored at −80° C. for purification.

Protein Purification.

Expressed full-length human GST-tagged PRMT1 protein was purified fromcell paste by glutathione sepharose affinity chromatography afterequilibration of the resin with 50 mM phosphate buffer, 200 mM NaCl, 5%glycerol, 5 mM (3-mercaptoethanol, pH7.8 (Buffer A). GST-tagged PRMT1was eluted with 50 mM Tris, 2 mM glutathione, pH 7.8, dialysed in bufferA and concentrated to 1 mg/mL. The purity of recovered protein was 73%.Reference: Wasilko, D. J. and S. E. Lee: “TIPS: titerless infected-cellspreservation and scale-up” Bioprocess J., 5 (2006), pp. 29-32.

Predicted Translations:

GST-tagged PRMT1 (SEQ ID NO.: 4)MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDENLYFQGGNSDYKDDDDKMAAAEAANCIMENFVATLANGMSLQPPLEEVSCGQAESSEKPNAEDMTSKDYYFDSYAHFGIHEEMLKDEVRTLTYRNSMFHNRHLFKDKVVLDVGSGTGILCMFAAKAGARKVIGIECSSISDYAVKIVKANKLDHVVTIIKGKVEEVELPVEKVDIIISEWMGYCLFYESMLNTVLYARDKWLAPDGLIFPDRATLYVTAIEDRQYKDYKIHWWENVYGFDMSCIKDVAIKEPLVDVVDPKQLVTNACLIKEVDIYTVKVEDLTFTSPFCLQVKRNDYVHALVAYFNIEFTRCHKRTGFSTSPESPYTHWKQTVFYMEDYLTVKTGEEIFGTIGMRPNAKNNRDLDFTIDLDFKGQLCEL SCSTDYRMR

General Procedure for PRMT1 Enzyme Assays on Peptide Substrates.

The assays were all performed in a buffer consisting of 20 mM Bicine(pH=7.6), 1 mM TCEP, 0.005% BSG, and 0.002% Tween 20, prepared on theday of use. Compounds in 100% DMSO (1 ul) were spotted into apolypropylene 384-well V-bottom plates (Greiner) using a Platemate Plusoutfitted with a 384-channel head (Thermo Scientific). DMSO (1 ul) wasadded to Columns 11, 12, 23, 24, rows A-H for the maximum signal controland 1 ul of SAH, a known product and inhibitor of PRMT1, was added tocolumns 11, 12, 23, 24, rows I-P for the minimum signal control. Acocktail (40 ul) containing the PRMT1 enzyme was added by MultidropCombi (Thermo-Fisher). The compounds were allowed to incubate with PRMT1for 30 min at room temperature, then a cocktail (10 ul) containing SAMand peptide was added to initiate the reaction (final volume=51 ul). Thefinal concentrations of the components were as follows: PRMT1 was 0.5nM, ³H-SAM was 200 nM, non-radiolabeled SAM was 1.5 uM, peptide was 20nM, SAH in the minimum signal control wells was 1 mM, and the DMSOconcentration was 2%. The assays were stopped by the addition ofnon-radiolabeled SAM (10 ul) to a final concentration of 300 uM, whichdilutes the ³H-SAM to a level where its incorporation into the peptidesubstrate is no longer detectable. 50 ul of the reaction in the 384-wellpolypropylene plate was then transferred to a 384-well Flashplate andthe biotinylated peptides were allowed to bind to the streptavidinsurface for at least 1 hour before being washed once with 0.1% Tween20in a Biotek ELx405 plate washer. The plates were then read in aPerkinElmer TopCount plate reader to measure the quantity of ³H-labeledpeptide bound to the Flashplate surface, measured as disintegrations perminute (dpm) or alternatively, referred to as counts per minute (cpm).

% Inhibition Calculation

${\%\mspace{14mu}{inh}} = {100 - {( \frac{{dpm}_{cmpd} - {dpm}_{\min}}{{dpm}_{\max} - {dpm}_{\min}} ) \times 100}}$

Where dpm=disintegrations per minute, cmpd=signal in assay well, and minand max are the respective minimum and maximum signal controls.

Four-Parameter IC50 Fit

$Y = {{Bottom} + \frac{( {{Top}\mspace{14mu}{Bottom}} )}{( {1 + ( \frac{X}{{IC}_{50}} )^{{Hill}\mspace{14mu}{Coefficient}}} }}$

Where top and bottom are the normally allowed to float, but may be fixedat 100 or 0 respectively in a 3-parameter fit. The Hill Coefficientnormally allowed to float but may also be fixed at 1 in a 3-parameterfit. Y is the % inhibition and X is the compound concentration.

TABLE 2 Biochemical IC₅₀ Cmpd No. PRMT1 1 A 2 B 3 B For Table 2, “A”indicates an IC₅₀ ≦ 0.100 μM, “B” indicates an IC₅₀ of 0.101-1.00 μM,“C” indicates an IC₅₀ of 1.01-3.00 μM, “D” indicates an IC₅₀ of 3.01-10μM, and IC₅₀ ≧ 10.01 μM.RKO Methylation Assay

RKO adherent cells were purchased from ATCC (American Type CultureCollection), Manassas, Va., USA. DMEM/Glutamax medium,penicillin-streptomycin, heat inactivated fetal bovine serum, 0.05%trypsin and D-PBS were purchased from Life Technologies, Grand Island,N.Y., USA. Odyssey blocking buffer, 800CW goat anti-rabbit IgG (H+L)antibody, and Licor Odyssey infrared scanner were purchased from LicorBiosciences, Lincoln, Nebr., USA. Mono-methyl arginine antibody waspurchased from Cell Signaling Technology, Danvers, Mass., USA.

Methanol was purchased from VWR, Franklin, Mass., USA. 10% Tween 20 waspurchased from KPL, Inc., Gaithersburg, Md., USA. DRAQ5 was purchasedfrom Biostatus Limited, Leicestershire, UK.

RKO adherent cells were maintained in growth medium (DMEM/Glutamaxmedium supplemented with 10% v/v heat inactivated fetal bovine serum and100 units/mL penicillin-streptomycin) and cultured at 37° C. under 5%CO₂.

Cell Treatment, in Cell Western (ICW) for Detection of Mono-MethylArginine and DNA Content.

RKO cells were seeded in assay medium at a concentration of 20,000 cellsper mL to a poly-D-lysine coated 384 well culture plate (BD Biosciences356697) with 50 μL per well. Compound (100 nL) from a 96-well sourceplate was added directly to 384 well cell plate. Plates were incubatedat 37° C., 5% CO₂ for 72 hours. After three days of incubation, plateswere brought to room temperature outside of the incubator for tenminutes and blotted on paper towels to remove cell media. 50 μL of icecold 100% methanol was added directly to each well and incubated for 30min at room temperature. After 30 min, plates were transferred to aBiotek EL406 plate washer and washed 2 times with 100 μL per well ofwash buffer (IX PBS). Next 60 μL per well of Odyssey blocking buffer(Odyssey Buffer with 0.1% Tween 20 (v/v)) were added to each plate andincubated 1 hour at room temperature. Blocking buffer was removed and 20μL per well of primary antibody was added (mono-methyl arginine diluted1:200 in Odyssey buffer with 0.1% Tween 20 (v/v)) and plates wereincubated overnight (16 hours) at 4° C. Plates were washed 5 times with100 μL per well of wash buffer. Next 20 μL per well of secondaryantibody was added (1:200 800CW goat anti-rabbit IgG (H+L) antibody,1:1000 DRAQ5 (Biostatus limited) in Odyssey buffer with 0.1% Tween 20(v/v)) and incubated for 1 hour at room temperature. The plates werewashed 5 times with 100 μL per well wash buffer then 2 times with 100 μLper well of water. Plates were allowed to dry at room temperature thenimaged on the Licor Odyssey machine which measures integrated intensityat 700 nm and 800 nm wavelengths. Both 700 and 800 channels werescanned.

Calculations:

First, the ratio for each well was determined by:

$( \frac{{monomethyl}\mspace{14mu}{Arginine}\mspace{14mu} 800\mspace{14mu}{nm}\mspace{14mu}{value}}{{DRAQS}\mspace{14mu} 700\mspace{14mu}{nm}\mspace{14mu}{value}} )$

Each plate included fourteen control wells of DMSO only treatment(minimum activation) as well as fourteen control wells for maximumactivation treated with 20 μM of a reference compound. The average ofthe ratio values for each control type was calculated and used todetermine the percent activation for each test well in the plate.Reference compound was serially diluted three-fold in DMSO for a totalof nine test concentrations, beginning at 20 μM. Percent activation wasdetermined and EC₃₀ curves were generated using triplicate wells perconcentration of compound.

${{Percent}\mspace{14mu}{Activation}} = {100 - ( {( \frac{( {{Individual}\mspace{14mu}{Test}\mspace{14mu}{Sample}\mspace{14mu}{Ratio}} ) - ( {{Minimum}\mspace{14mu}{Activation}\mspace{14mu}{Ratio}} )}{( {{Maximum}\mspace{14mu}{Activation}\mspace{14mu}{Ratio}} ) - ( {{Minimum}\mspace{14mu}{Activation}\mspace{14mu}{Ratio}} )} )*100} )}$

TABLE 3 In Cell Western data Cmpd No. EC₃₀ 1 B 2 C For Table 3, “A”indicates an EC₃₀ ≦ 3.00 μM, “B” indicates an EC₃₀ of 3.01-12.00 μM, and“C” indicates an EC₃₀ ≧ 12.01 μM.

Other Embodiments

The foregoing has been a description of certain non-limiting embodimentsof the invention. Those of ordinary skill in the art will appreciatethat various changes and modifications to this description may be madewithout departing from the spirit or scope of the present invention, asdefined in the following claims.

What is claimed is:
 1. A method of treating a PRMT1-mediated disorder,the method comprising administering to a subject in need thereof atherapeutically effective amount of a compound selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.
 2. The method of claim 1,wherein the PRMT1-mediated disorder is a proliferative disorder.
 3. Themethod of claim 2, wherein the proliferative disorder is cancer.
 4. Themethod of claim 1, wherein the PRMT1-mediated disorder is a neurologicaldisorder.
 5. The method of claim 4, wherein the neurological disorder isamyotrophic lateral sclerosis.
 6. The method of claim 1, wherein thePRMT1-mediated disorder is a muscular dystrophy.
 7. The method of claim1, wherein the PRMT1-mediated disorder is an autoimmune disorder.
 8. Themethod of claim 1, wherein the PRMT1-mediated disorder is a vasculardisorder.
 9. The method of claim 1, wherein the PRMT1-mediated disorderis a metabolic disorder.